Patent Application
20240269348
Brain injury can lead to a loss of neurons. While much work has been done to enhance neuronal recovery after brain injury, these efforts have not led to sustained improvements.
Provided herein, inter alia, is a method for obtaining an astrocyte from a gingiva-derived mesenchymal stem cell (GDMSC) and methods of using the same in a system comprising a biocompatible construct, e.g., as disclosed herein. Also provided herein are uses of a system comprising a plurality of astrocytes obtained from GDMSCs for, e.g., promoting cell migration or treating a neurodegenerative disorder or a neurological disorder, e.g., as described herein.
This disclosure provides, a method of obtaining at least one astrocyte, the method comprising: (a) providing at least one gingiva-derived mesenchymal stem cell (GDMSC), and (b) contacting at least one GDMSC with: (i) a first serum-free medium comprising N-2 supplement, basic fibroblast growth factor 2 (bFGF) and epidermal growth factor (EGF); and (ii) a second serum-free medium comprising: dibutyryl cyclic adenosine monophosphate (dbcAMP), IBMX (3-Isobutyl-1-methylxanthine), neuregulin, and platelet-derived growth factor (PDGF), wherein a GDMSC is contacted with a first medium and/or a second medium under conditions sufficient to induce differentiation of the GDMSC into an astrocyte.
In some embodiments, a GDMSC is obtained from a subject, e.g., a human.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 75% cells are cranial neural crest derived mesenchymal stem cells (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%). In some embodiments, a GDMSC is obtained from a population of cells in which no more than 25% cells are mesoderm derived mesenchymal stem cells (e.g. no more than 20%, 15%, 10% 5%, or 1%). In some embodiments, a GDMSC is obtained from a population of cells in which at least 75% cells are cranial neural crest derived mesenchymal stem cells and no more than 25% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a neural crest derived mesenchymal stem cell has an increased ability to differentiate into neuronal cells as compared to mesoderm derived mesenchymal stem cells. In some embodiments, a neural crest derived mesenchymal stem cell has an increased immunomodulatory capacity compared to mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is contacted with a first medium for about 72 hours.
In some embodiments, a GDMSC is contacted with a second medium for about 72 hours.
In some embodiments, a first medium and/or a second medium further comprises one or more additional components. In some embodiments, one or more additional components comprises: an antibiotic, an amino acid supplement, an anti-fungal, or a combination thereof.
In some embodiments, a first medium comprises about 20 ng/ml of N-2 supplement, about 20 ng/ml basic fibroblast growth factor 2 (bFGF) and about 20 ng/ml epidermal growth factor (EGF).
In some embodiments, a second medium comprises about 0.5 to 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP), about 0.5 to 1 mM IBMX (3-Isobutyl-1-methylxanthine), about 50 ng/ml to 100 ng/ml of neuregulin, and about 1 ng/ml to 5 ng/ml of platelet-derived growth factor (PDGF).
In some embodiments, a GDMSC disclosed herein comprises one or more of the following characteristics:
In some embodiments, a GDMSC disclosed herein comprises any one of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises any two of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises any three of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises any four of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises any five of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises any six of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises any seven of (i) to (viii).
In some embodiments, a GDMSC disclosed herein comprises all of (i) to (viii).
In some embodiments, of any of the methods disclosed herein, the amount of time required to obtain an astrocyte is reduced relative to a comparator. In some embodiments, a comparator comprises an otherwise similar method using induced pluripotent stem cells (iPSC) to obtain an astrocyte.
In some embodiments, the amount of time required to obtain an astrocyte is less than 1 week, less than 6 days, less than 5 days, or less than 4 days.
In some embodiments, of any of the methods disclosed herein, an astrocyte obtained from a method disclosed herein comprises one or more of the following characteristics:
In some embodiments, an astrocyte obtained from a method disclosed herein comprises any one of (i)-(vi).
In some embodiments, an astrocyte obtained from a method disclosed herein comprises any two of (i)-(vi).
In some embodiments, an astrocyte obtained from a method disclosed herein comprises any three of (i)-(vi).
In some embodiments, an astrocyte obtained from a method disclosed herein comprises any four of (i)-(vi).
In some embodiments, an astrocyte obtained from a method disclosed herein comprises any five of (i)-(vi).
In some embodiments, an astrocyte obtained from a method disclosed herein comprises all of (i)-(vi).
In some embodiments, a bundle of astrocytes comprises a structure similar or substantially similar to a structure of an astrocyte in an endogenous rostral migratory stream.
In some embodiments, a bundle of astrocytes comprises a function similar to or substantially similar to a function of an astrocyte in an endogenous rostral migratory stream.
Also provided herein is a reaction mixture comprising at least one GDMSC and a serum-free medium comprising N-2 supplement, basic fibroblast growth factor 2 (bFGF) and epidermal growth factor (EGF).
In some embodiments, a reaction mixture further comprises one or more additional components. In some embodiments, one or more additional components comprises: an antibiotic, an amino acid supplement, an anti-fungal, or a combination thereof.
This disclosure further provides, a reaction mixture comprising at least one GDMSC and a medium comprising: dibutyryl cyclic adenosine monophosphate (dbcAMP), IBMX (3-Isobutyl-1-methylxanthine), neuregulin, and platelet-derived growth factor (PDGF).
In some embodiments, a reaction mixture further comprises one or more additional components. In some embodiments, one or more additional components comprises: an antibiotic, an amino acid supplement, an anti-fungal, or a combination thereof.
In some embodiments, of a reaction mixtures disclosed herein, a reaction mixture is maintained under conditions sufficient to obtain an astrocyte.
In some embodiments, of a reaction mixtures disclosed herein, an astrocyte obtained from a reaction mixture comprises one or more of the following characteristics:
In some embodiments, of a reaction mixtures disclosed herein, a bundle of astrocytes comprises a structure similar or substantially similar to a structure of an astrocyte in an endogenous rostral migratory stream.
In some embodiments, of a reaction mixtures disclosed herein, a bundle of astrocytes comprises a function similar to or substantially similar to a function of an astrocyte in an endogenous rostral migratory stream.
Further provided herein is a system, comprising: (a) a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC); and (b) a biocompatible construct comprising a matrix and having a first end, a second end and a body.
In some embodiments, a plurality of astrocytes used in a system disclosed herein is obtained according to a method disclosed herein.
In some embodiments, a system disclosed herein further comprises a Slit-Robo entity. In some embodiments, a Slit-Robo entity is or comprises an agent that promotes activation and/or signaling from a Slit-Robo pathway.
In some embodiments, a system disclosed herein is characterized in that when implanted into an organism, it promotes migration of one or more cells compared to a comparator. In some embodiments, a comparator comprises an otherwise similar system without a plurality of astrocytes; or without a plurality of astrocytes derived from at least one GDMSC.
In some embodiments, one or more migrating cells comprises one or more endogenous host cells. In some embodiments, an endogenous host cell comprises a neural precursor cell, a neuroblast, a neuron, a progenitor cell, a glial cell, an astrocyte, and/or an endothelial cell.
In some embodiments, one or more migrating cells comprises: one or more cells provided in the system; or one or more cells derived from, differentiated from or a progenitor of a cell provided in the system.
In some embodiments, migration of one or more cells occurs within the system, throughout the system, out of the system or into the system.
In some embodiments, migration of one or more cells occurs to the site of implantation, or away from the site of implantation.
In some embodiments of a system disclosed herein, a matrix of a biocompatible construct comprises an inner surface and an outer surface.
In some embodiments, an inner surface of the biocompatible construct defines a luminal core.
In some embodiments, an outer surface of the biocompatible construct comprises at least one hydrogel.
In some embodiments, a hydrogel comprises one or more of agarose, hyaluronic acid, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose.
In some embodiments, a hydrogel comprises hyaluronic acid. In some embodiments, a hyaluronic acid is or comprises methacrylated HA (MeHA).
In some embodiments, a hydrogel is or comprises agarose. In some embodiments, an agarose is at about 0.25-30%, about 0.25%-3%, about 0.5%-3%, about 1-20%, about 1.5-10%, about 2-9%, about 2.5-8%, or about 3-7%. In some embodiments, an agarose is at about 3%.
In some embodiments, an inner surface of the biocompatible construct comprises one or more extracellular matrix (ECM) components.
In some embodiments, an ECM component comprises collagen, laminin, fibronectin, hyaluronic acid, or a combination thereof.
In some embodiments, an ECM comprises collagen. In some embodiments, a collagen is at a concentration of about 0.1-10 mg/ml or 0.1-9 mg/ml. In some embodiments, a collagen is at a concentration of about 1 mg/ml.
In some embodiments, a plurality of astrocytes is seeded at least once in a system disclosed herein.
In some embodiments, a plurality of astrocytes is seeded twice in a system disclosed herein.
In some embodiments, a plurality of astrocytes is seeded at a cell density of about 0.1 million cells/ml to about 10 million cells/ml. In some embodiments, a plurality of astrocytes is seeded at a cell density of about 1 million cells/ml.
In some embodiments, a plurality of astrocytes comprises at least 500 cells, at least 1000 cells, at least 5000 cells, at least 10,000 cells, at least 15,000 cells, at least 20,000 cells, at least 40,000 cells, at least 80,000 cells, at least 100,000 cells, or at least 500,000 cells.
In some embodiments, a system disclosed herein further comprises one or more additional cells or components. In some embodiments, an additional cell is not an astrocyte, e.g., as described herein. In some embodiments, an additional cell is or comprises an endothelial cell. In some embodiments, a one or more additional cells or components induces vascularization.
In some embodiments, an additional cell or component is introduced into the system: (i) concurrently with, before or after seeding of the plurality of astrocytes; (ii) during formation of an astrocyte bundle by the plurality of astrocytes; and/or (ii) after formation of an astrocyte bundle by the plurality of astrocytes.
Disclosed herein is a method of manufacturing a system comprising: (a) a biocompatible construct comprising a matrix and having a first end, a second end and a body; and (b) associating the biocompatible construct with a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC).
In some embodiments, a method of manufacturing comprises maintaining a system under conditions that promotes growth of at least one astrocyte in a plurality of astrocytes.
In some embodiments, a method of manufacturing comprises maintaining a system under conditions that maintain viability of at least one astrocyte in a plurality of astrocytes.
In some embodiments, a method of manufacturing comprises forming an aggregate of at least a portion of the plurality of astrocytes.
In some embodiments, a system in a method of manufacturing disclosed herein comprises any one of a system disclosed herein.
Provided herein is a method of promoting cell migration, comprising:
In some embodiments, a method of promoting cell migration comprises implanting a system disclosed herein in a subject. In some embodiments, a system is implanted: (i) at, near or within a brain lesion in a subject; (ii) at, near or within an area in the brain with insufficient neurons or neuronal connections, or with damaged neurons or neuronal connections; (iii) at, near or within a subventricular zone, an endogenous rostral migratory stream or a neurogenic niche; and/or (iv) at, near or within a region of a brain affected by a brain injury, a neurodegenerative disease or disorder, or a neurodevelopmental disease or disorder.
In some embodiments, one or more migrating cells comprises one or more endogenous host cells.
In some embodiments, an endogenous host cell comprises a neural precursor cell, a neuroblast, a neuron, a progenitor cell, a glial cell, an astrocyte, and/or an endothelial cell.
In some embodiments, one or more migrating cells comprises: one or more cells provided in a system, or one or more cells derived from, differentiated from or a progenitor of a cell provided in a system.
In some embodiments, migration of one or more cells occurs within a system, throughout a system, out of a system or into a system.
In some embodiments, migration of one or more cells occurs a site of implantation, or away from a site of implantation.
This disclosure also provides a method of treating a neurodegenerative disorder or a neurological disorder in a subject, comprising providing to a subject a system comprising: (a) a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC); and (b) a biocompatible construct comprising a matrix and having a first end, a second end and a body.
Also provided herein is a method of ameliorating one or more symptoms of a neurodegenerative disorder or a neurological disorder in a subject, comprising providing to a subject a system comprising: (a) a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC); and (b) a biocompatible construct comprising a matrix and having a first end, a second end and a body.
In some embodiments, a neurodegenerative disorder comprises a disorder with injury or degeneration to one or more neurons.
In some embodiments, a neurodegenerative disorder comprises brain injury or spinal cord injury.
In some embodiments, brain injury comprises acute brain injury, degenerative brain injury, traumatic brain injury (TBI), or chronic brain injury.
In some embodiments, a neurological disorder comprises: Parkinson's, Alzheimer's, Huntington's, prion disease, motor neuron disease, spinocerebellar ataxia, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), encephalitis, epilepsy, head and brain malformations, or hydrocephalus.
In some embodiments, a subject is a human.
In some embodiments of a method disclosed herein, a system disclosed herein is implanted into a subject. In some embodiments, a system is implanted into a brain of a subject.
In some embodiments, a system is implanted: (i) at, near or within a brain lesion in a subject; (ii) at, near or within an area in a brain with insufficient neurons or neuronal connections, or with damaged neurons or neuronal connections; (iii) at, near or within a subventricular zone, an endogenous rostral migratory stream or a neurogenic niche; and/or (iv) at, near or within a region of a brain affected by a brain injury, a neurodegenerative disease or disorder, or a neurodevelopmental disease or disorder.
In some embodiments of a method disclosed herein, a system is characterized in that when implanted into an organism it ameliorates or reduces severity of one or more symptoms of a disorder.
In some embodiments of a method disclosed herein, a system is characterized in that when implanted into an organism it promotes migration of one or more cells.
In some embodiments, migration of one or more cells is compared to a comparator. In some embodiments, a comparator comprises an organism implanted with an otherwise similar system without a plurality of astrocytes; or without a plurality of astrocytes derived from at least one GDMSC.
In some embodiments, one or more migrating cells comprises one or more endogenous host cells.
In some embodiments, an endogenous host cell comprises a neural precursor cell, a neuroblast, a neuron, a progenitor cell, a glial cell, an astrocyte, and/or an endothelial cell.
In some embodiments, one or more migrating cells comprises: one or more cells provided in a system; or one or more cells derived from, differentiated from or a progenitor of a cell provided in a system.
In some embodiments, migration of one or more cells occurs within a system, throughout a system, out of a system or into a system.
In some embodiments, migration of one or more cells occurs to a site of implantation, or away from a site of implantation.
Also provided herein is a kit comprising, a system comprising: (a) a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC); (b) a biocompatible construct comprising a matrix and having a first end, a second end and a body; and (c) instructions for using the same.
In some embodiments, a plurality of astrocytes is obtained according to a method disclosed herein.
As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
As used herein, “synapse” refers to a junction between a neuron and another cell, across which chemical communication flows.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).
In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and/or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated In some embodiments, an engineered cell is one that has been manipulated so that it contains and/or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and/or a particular form thereof) in an altered amount and/or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorder, and/or condition. In some embodiments, a subject is susceptible to a disease, disorder, and/or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, and/or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, and/or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
The term “biocompatible construct”, as used herein, refers to a construct comprising a matrix that does not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo (e.g., over a period of time). In certain embodiments, a biocompatible construct is not toxic to cells (e.g., over a period of time). In certain embodiments, a biocompatible construct does not induce significant inflammation or other immune response, or other adverse effects when placed or implanted into a subject (e.g., over a period of time). In some embodiments, a matrix of a biocompatible construct comprises a scaffold. In some embodiments, a matrix of a biocompatible construct comprises an inner surface, an outer surface or both an inner surface and an outer surface. In some embodiments, an inner surface of a matrix of a biocompatible construct defines a luminal core. In some embodiments, an inner surface of a matrix of a biocompatible construct comprises a component disclosed herein. In some embodiments, an outer surface of a matrix of a biocompatible construct comprises a component disclosed herein. In some embodiments, a biocompatible construct comprises one or more additional components.
The term “astrocyte,” as used herein refers to a non-neuronal cell which supports proliferation, differentiation, signaling and/or homeostasis of a neuronal cell. In some embodiments, an astrocyte is present in a central nervous system of an organism. In some embodiments, an astrocyte is obtained from a central nervous system of an organism. In some embodiments, an astrocyte is derived from a stem cell, e.g., a mesenchymal stem cell, e.g., a GDMSC. In some embodiments, an astrocyte has one or more of the following characteristics: (i) expresses GFAP, Ezrin, Robo2, S-100-beta, glutamine synthetase (GS) and/or Glutamate Aspartate Transporter (GLAST); (ii) expresses pyruvate carboxylase (PC) and/or Glutamate transporter-1 (GLT-1); (iii) has low or no expression of an endothelial marker, e.g., CD31; (iv) is capable of self-assembly into a bundle of longitudinally-aligned astrocytes with bipolar or multipolar processes, (v) is capable of forming a bundle of astrocytes; and/or (vi) has a morphology of an astrocyte. In some embodiments, an astrocyte is capable of self-assembly into a bundle of longitudinally aligned astrocytes with bipolar (e.g., bidirectional) processes. In some embodiments, an astrocyte has a “star-like” morphology, e.g., as observed in protoplasmic astrocytes. In some embodiments, an astrocyte has a linear morphology, e.g., as observed in fibrous astrocytes. In some embodiments, an astrocyte has any one of characteristics (i) to (vi). In some embodiments, an astrocyte has any two of characteristics (i) to (vi). In some embodiments, an astrocyte has any three of characteristics (i) to (vi). In some embodiments, an astrocyte has any four of characteristics (i) to (vi). In some embodiments, an astrocyte has any five of characteristics (i) to (vi). In some embodiments, an astrocyte has all of characteristics (i) to (vi).
The present disclosure identifies certain challenges that can be associated with regeneration of neurons upon injury to the brain. For example, the present disclosure identifies certain problems that can be encountered with inducing neuronal regeneration and/or migration of nascent neurons to a site of injury.
Brain injury can cause loss of neurons and endogenous neurogenesis is upregulated in the subventricular zone (SVZ) following brain injury (16-18). In adults, during neurogenesis neural precursor cells (NPCs) in the SVZ can differentiate into neuroblasts and migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) where they mature into interneurons and integrate into existing circuitry (3-6). Increased neurogenesis has been reported in the rodent SVZ following multiple experimental models of acquired brain injury. Following brain injury, newly formed NPCs can mature into neuroblasts, divert from the SVZ/RMS, and migrate toward injured brain regions (27, 28, 34, 35)(
Neural tissue engineering has introduced the possibility of developing customized therapies to enhance neuronal regeneration following traumatic brain injury. A variety of biomaterial and tissue engineering technologies have been developed to enhance the neurogenic potential of the SVZ and redirect the migration of SVZ neuroblasts to neuron-deficient brain regions following various experimental brain injuries. The evidence from tissue engineering techniques, along with that of pharmacological and genetic approaches, collectively demonstrates that experimental intervention to enhance the brain's intrinsic repair mechanism to replace lost or damaged neurons with endogenous SVZ NPCs can improve recovery after acquired brain injury. However, while promising, these interventions have thus far only afforded transient re-direction of neuroblasts.
Among other things, the present disclosure provides technologies for improving functional recovery upon brain injury. For example, this disclosure provides novel methods for generating astrocytes from gingiva-derived mesenchymal stem cells (GDMSC), a source of mesenchymal stem cells that is easily accessible, and more homogenous, e.g., as compared to oral mucosal stem cell (OMSC). In some embodiments, a GDMSC is obtained from a population of cells having at least 75% (e.g., at least 80%, 85%, 90%, 95%, 99%) neural crest derived stem cells. In some embodiments, an astrocyte obtained from gingiva-derived mesenchymal stem cells (GDMSC), can be used in a system disclosed herein (e.g., a TE-RMS) to promote neuronal regeneration, proliferation and/or migration.
In some embodiments, a system disclosed herein, e.g., a TE-RMS, promotes migration of a cell (e.g., an endogenous cell, e.g., an NPC or a neuroblast) through a TE-RMS to a site of injury. In some embodiments, a system disclosed herein, e.g., a TE-RMS, promotes or supports differentiation of a precursor cell, e.g., an NPC, into a neuroblast. Without wishing to be bound by theory, the present disclosure proposes that a system disclosed herein, e.g., a TE-RMS, redirects migration of endogenous neuroblasts out of the RMS and into distal lesions, to provide sustained delivery to replace lost neurons and improve functional recovery. In some embodiments, a system disclosed herein, e.g., a TE-RMS disclosed herein, emulates a brain's known method for transporting neuroblasts to a distal area for neuronal replacement. In some embodiments, a sustained influx of new neurons is likely required for functional improvements across a spectrum of brain injury severities.
Among other things, this disclosure provides a method of obtaining an astrocyte from a gingiva-derived mesenchymal stem cell (GDMSC). In some embodiments, a method of obtaining an astrocyte from a GDMSC comprises (a) providing at least one gingiva-derived mesenchymal stem cell (GDMSC), and (b) contacting at least one GDMSC with certain serum-free media, under conditions sufficient to induce differentiation of a GDMSC into an astrocyte.
In some embodiments, a method of obtaining an astrocyte disclosed herein comprises contacting a GDMSC with a first serum-free medium comprising N-2 supplement, basic fibroblast growth factor 2 (bFGF) and epidermal growth factor (EGF). In some embodiments, a method disclosed herein comprises contacting a GDMSC with a further, e.g., a second, particular serum-free medium.
In some embodiments, a method of obtaining an astrocyte disclosed herein comprises contacting a GDMSC with a second serum-free medium comprising: dibutyryl cyclic adenosine monophosphate (dbcAMP), IBMX (3-Isobutyl-1-methylxanthine), neuregulin, and platelet-derived growth factor (PDGF). In some embodiments, a method disclosed herein comprises contacting a GDMSC with a different, e.g., a first, serum-free medium.
In some embodiments, a first serum-free medium comprises about 5-40 ng/ml, about 5-35 ng/ml, about 5-30 ng/ml, about 5-25 ng/ml, about, 5-20, ng/ml, about 10-40 ng/ml, about 15-30 ng/ml, about 15-25 ng/ml, about 15-20 ng/ml of N-2 supplement. In some embodiments, a first serum-free medium comprises about 20 ng/ml of N-2 supplement.
In some embodiments, a first serum-free medium comprises about 5-40 ng/ml, about 5-35 ng/ml, about 5-30 ng/ml, about 5-25 ng/ml, about, 5-20, ng/ml, about 10-40 ng/ml, about 15-30 ng/ml, about 15-25 ng/ml, about 15-20 ng/ml of basic fibroblast growth factor 2 (bFGF). In some embodiments, a first serum-free medium comprises about 20 ng/ml of basic fibroblast growth factor 2 (bFGF).
In some embodiments, a first serum-free medium comprises about 5-40 ng/ml, about 5-35 ng/ml, about 5-30 ng/ml, about 5-25 ng/ml, about, 5-20, ng/ml, about 10-40 ng/ml, about 15-30 ng/ml, about 15-25 ng/ml, about 15-20 ng/ml of epidermal growth factor (EGF). In some embodiments, a first serum-free medium comprises about 20 ng/ml of epidermal growth factor (EGF).
In some embodiments, a first serum-free medium comprises about 20 ng/ml of N-2 supplement, about 20 ng/ml basic fibroblast growth factor 2 (bFGF) and about 20 ng/ml epidermal growth factor (EGF).
In some embodiments, a method of obtaining an astrocyte disclosed herein comprises contacting a GDMSC with a first serum-free medium comprising N-2 supplement, basic fibroblast growth factor 2 (bFGF) and epidermal growth factor (EGF) and a second serum-free medium comprising: dibutyryl cyclic adenosine monophosphate (dbcAMP), IBMX (3-Isobutyl-1-methylxanthine), neuregulin, and platelet-derived growth factor (PDGF).
In some embodiments, a second medium comprises about 0.5 to 1 mM, about 0.6 to 1 mM, about 0.7 to 1 mM, about 0.8 to 1 mM, about 0.9 to 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP). In some embodiments, a second medium comprises about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP).
In some embodiments, a second medium comprises about 0.5 to 1 mM, about 0.6 to 1 mM, about 0.7 to 1 mM, about 0.8 to 1 mM, about 0.9 to 1 mM IBMX (3-Isobutyl-1-methylxanthine). In some embodiments, a second medium comprises about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 mM IBMX (3-Isobutyl-1-methylxanthine).
In some embodiments, a second medium comprises about 50 ng/ml to 100 ng/ml, about 60 ng/ml to 100 ng/ml, about 70 ng/ml to 100 ng/ml, about 80 ng/ml to 100 ng/ml, about 90 ng/ml to 100 ng/ml, about 95 ng/ml to 100 ng/ml of neuregulin. In some embodiments, a second medium comprises about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml of neuregulin.
In some embodiments, a second medium comprises about 1 ng/ml to 5 ng/ml, about 1.5 to 5 ng/ml, about 2 to 5 ng/ml, about 2.5 to 5 ng/ml, about 3 to 5 ng/ml, about 3.5 to 5 ng/ml, about 4 to 5 ng/ml, about 4.5 to 5 ng/ml of platelet-derived growth factor (PDGF). In some embodiments, a second medium comprises about 1 ng/ml, about 1.5 ng/ml, about 2 ng/ml, about 2.5 ng/ml, about 3 ng/ml, about 3.5 ng/ml, about 4 ng/ml, about 4.5 ng/ml, about 5 ng/ml of platelet-derived growth factor (PDGF).
In some embodiments, a second medium comprises about 0.5 to 1 mM dibutyryl cyclic adenosine monophosphate (dbcAMP), about 0.5 to 1 mM IBMX (3-Isobutyl-1-methylxanthine), about 50 ng/ml to 100 ng/ml of neuregulin, and about 1 ng/ml to 5 ng/ml of platelet-derived growth factor (PDGF).
In some embodiments, a first medium and/or a second medium further comprises one or more additional components. In some embodiments, one or more additional components comprises: an antibiotic, an amino acid supplement, an anti-fungal, or a combination thereof.
In some embodiments, a GDMSC is contacted with a first medium for about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours or about 80 hours. In some embodiments, a GDMSC is contacted with a first medium for about 65-80 hours, for about 65-75 hours, for about 68-75 hours, or for about 70-75 hours. In some embodiments, a GDMSC is contacted with a first medium for about 72 hours.
In some embodiments, a GDMSC is contacted with a second medium for about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours, about 75 hours, about 76 hours, about 77 hours, about 78 hours, about 79 hours or about 80 hours. In some embodiments, a GDMSC is contacted with a second medium for about 65-80 hours, for about 65-75 hours, for about 68-75 hours, or for about 70-75 hours. In some embodiments, a GDMSC is contacted with a second medium for about 72 hours.
In some embodiments of a method of obtaining an astrocyte described herein, the amount of time required to obtain an astrocyte is reduced relative to a comparator. In some embodiments, a comparator comprises an otherwise similar method using induced pluripotent stem cells (iPSC) to obtain an astrocyte.
In some embodiments of a method of obtaining an astrocyte described herein, the amount of time required to obtain an astrocyte the amount of time required to obtain an astrocyte is less than 1 week, less than 6 days, less than 5 days, or less than 4 days. In some embodiments, the amount of time required to obtain an astrocyte is less than 1 week. In some embodiments, the amount of time required to obtain an astrocyte is less than 6 days. In some embodiments, the amount of time required to obtain an astrocyte is less than 5 days, In some embodiments, the amount of time required to obtain an astrocyte is less than 4 days.
In some embodiments, the amount of time required to obtain an astrocyte is about 1 week. In some embodiments, the amount of time required to obtain an astrocyte is about 6 days. In some embodiments, the amount of time required to obtain an astrocyte is about 5 days. In some embodiments, the amount of time required to obtain an astrocyte is about 4 days.
In some embodiments, a method of obtaining an astrocyte described herein comprises culturing a GDMSC at about 37 degrees Celsius. In some embodiments, a method of obtaining an astrocyte described herein comprises culturing a GDMSC in about 5% CO2.
In some embodiments, a method of obtaining an astrocyte described herein further comprises culturing an astrocyte obtained from a method disclosed herein under conditions sufficient to maintain viability and/or induce proliferation of an astrocyte. In some embodiments, an astrocyte obtained from a method disclosed herein is cultured in media, e.g., DMEM/F12 medium, supplemented with serum, e.g., 10% FBS, and an antibiotic, e.g., 1% Penicillin-Streptomycin.
In some embodiments, an astrocyte obtained from a method disclosed herein comprises one or more characteristics of an astrocyte, e.g., as described herein. In some embodiments, an astrocyte has one or more of the following characteristics: (i) expresses GFAP, Ezrin, Robo2, S-100-beta, glutamine synthetase (GS) and/or GLutamate ASpartate Transporter (GLAST); (ii) expresses pyruvate carboxylase (PC) and/or Glutamate transporter-1 (GLT-1); (iii) has low or no expression of an endothelial marker, e.g., CD31; (iv) is capable of self-assembly into a bundle of longitudinally-aligned astrocytes with bipolar or multipolar processes, (v) is capable of forming a bundle of astrocytes; and/or (vi) has a morphology of an astrocyte. In some embodiments, an astrocyte is capable of self-assembly into a bundle of longitudinally aligned astrocytes with bidirectional (e.g., bipolar) or multidirectional processes. In some embodiments, an astrocyte has a “star-like” morphology, e.g., as observed in protoplasmic astrocytes. In some embodiments, an astrocyte has a linear morphology, e.g., as observed in fibrous astrocytes.
In some embodiments, an astrocyte obtained from a method disclosed herein is cultured for about 1-20 passages, about 2-15 passages, or about 4-10 passages.
In some embodiments, a culture of an astrocyte obtained from a method disclosed herein is subjected to perturbation, e.g., mechanical perturbation. In some embodiments, mechanical perturbation of a culture of an astrocyte obtained from a method disclosed herein allows for purification of an astrocyte population. In some embodiments, a culture of an astrocyte is mechanically perturbed prior to trypisinization.
Mesenchymal stem cells derived from human gingiva were first described by Zhang Q Z et al., (2009) Journal of Immunology 183:7787-7798, the entire contents of which are hereby incorporated by reference. Such gingiva-derived mesenchymal stem cells (GDMSC) comprise stem-cell like properties and immunomodulatory properties. Additional properties of GDMSCs such as GDMSCs comprising a population of cells that is primarily derived from cranial neural crest stem cells were previously disclosed by Xu X. et al., (2013) J. Dent Res 92(9): 825-832, the entire contents of which are hereby incorporated by reference.
In part, the present disclosure encompasses the surprising finding that GDMSCs can differentiate into astrocytes. GDMSCs possess several characteristics that are not shared by other mesenchymal stem cells (MSCs). For example, GDMSCs exhibit a proliferative capacity beyond that of other MSCs, including bone marrow-derived MSCs and umbilical cord-derived MSCs, and are also capable of potent immunomodulatory effects. Further, GDMSCs exhibit the potential to differentiate into a variety of cell types including neurons, keratinocytes, and endothelial cells. See Kim et al., (2021) Gingiva-Derived Mesenchymal Stem Cells: Potential Application in Tissue Engineering and Regenerative Medicine—A Comprehensive Review, Frontiers in Immunology, 12: 667221. As is shown in the examples below, the present disclosure describes, inter alia, the creation of astrocytes and their use in new tissue engineered constructs.
Furthermore, Zhang Q Z et al., (2012) disclosed that gingiva, from which GDMSCs are derived, is a unique microenvironment fueled by food residues, microbial flora and saliva, and is recognized for, e.g., its sensitivity to inflammation, fibrosis response and proneness to drug-induced overgrowth (See Zhang Q Z et al., (2012) J Dent Res 91(11): 1011-1018).
Accordingly, in some embodiments, a GDMSC disclosed herein is obtained from a population of cells that is relatively homogenous, e.g., as compared to oral mucosal stem cells (OMSC).
In some embodiments, a GDMSC is obtained from a population of cells in which at least 75% cells, at least 76% cells, at least 77% cells, at least 78% cells, at least 79% cells, at least 80% cells, at least 81% cells, at least 82% cells, at least 83% cells, at least 85% cells, at least 86% cells, at least 87% cells, at least 88% cells, at least 89% cells, at least 90% cells, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% cells are cranial neural crest derived mesenchymal stem cells. In some embodiments, about 75% to 100%, or about 80% to 100%, about 85% to 100%, about 90% to 100%, or about 91% to 100%, about 92% to 100%, about 93% to 100%, about 94% to 100%, about 95% to 100%, about 96% to 100%, about 97% to 100%, about 98% to 100%, about 99% to 100%, about 75% to 99%, about 75% to 98%, about 75% to 97%, about 75% to 96% about 75% to 95%, about 75% to 94%, about 75% to 93%, about 75% to 92%, or about 75% to 91% cells, about 75% to 90%, about 75% to 85%, or about 75% to 80% cells are cranial neural crest derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2% or no more than 1% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 75% cells are cranial neural crest derived mesenchymal stem cells and no more than 25% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 80% cells are cranial neural crest derived mesenchymal stem cells and no more than 20% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 85% cells are cranial neural crest derived mesenchymal stem cells and no more than 15% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 90% cells are cranial neural crest derived mesenchymal stem cells and no more than 10% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 95% cells are cranial neural crest derived mesenchymal stem cells and no more than 5% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a GDMSC is obtained from a population of cells in which at least 99% cells are cranial neural crest derived mesenchymal stem cells and no more than 1% cells are mesoderm derived mesenchymal stem cells.
In some embodiments, a neural crest derived mesenchymal stem cell has an increased ability to differentiate into neuronal cells as compared to mesoderm derived mesenchymal stem cells.
In some embodiments, a neural crest derived mesenchymal stem cell has an increased immunomodulatory capacity compared to mesoderm derived mesenchymal stem cells. In some embodiments, an immunomodulatory capacity comprises: (i) increase in number and/or activity of T regulatory cells; (ii) decrease in number and/or activity of inflammatory T cells such as Th17 cells; (iii) increase in number and/or activity of cytotoxic T cells, or a combination thereof.
In some embodiments, a GDMSC comprises one or more of the following characteristics:
In some embodiments, a GDMSC comprises any one of (i)-(ix).
In some embodiments, a GDMSC comprises any two of (i)-(ix).
In some embodiments, a GDMSC comprises any three of (i)-(ix).
In some embodiments, a GDMSC comprises any four of (i)-(ix).
In some embodiments, a GDMSC comprises any five of (i)-(ix).
In some embodiments, a GDMSC comprises any six of (i)-(ix).
In some embodiments, a GDMSC comprises any seven of (i)-(ix).
In some embodiments, a GDMSC comprises any eight of (i)-(ix).
In some embodiments, a GDMSC comprises all of (i)-(ix).
Astrocytes are cells with distinctive morphological and functional characteristics that depend on the location of the cells in the central nervous system or brain. In general, astrocytes have a regulatory role and have been shown to play a role, e.g., in neurogenesis and synaptogenesis; controlling blood-brain barrier permeability; and maintaining extracellular homeostasis (see Siracusa R. et al. (2019) Front. Pharmacol. 10:1114, the entire contents of which are hereby incorporated by reference). One of the functions of astrocytes is in synaptic regulation, e.g., in the formation and maturation of synapses; and/or in the maintenance, pruning and remodeling of synapses during development, aging and/or diseases (see Matias I., et al (2019) Front. Aging Neurosci. 11:59, the entire contents of which are hereby incorporated by reference). Due in part to the heterogeneity of astrocytes, these cells are also implicated in a wide array of neuropathologies.
Exemplary astrocytes, e.g., as disclosed herein, include protoplasmic astrocytes, fibrous astrocytes, varicose astrocytes, interlaminar projection astrocytes, radial glial cells, Bergmann glia and Muller glia. In some embodiments, an astrocyte has one or more of the following characteristics: (i) expresses GFAP, Ezrin, Robo2, S-100-beta, glutamine synthetase (GS) and/or GLutamate ASpartate Transporter (GLAST); (ii) expresses pyruvate carboxylase (PC) and/or Glutamate transporter-1 (GLT-1); (iii) has low or no expression of an endothelial marker, e.g., CD31; (iv) is capable of self-assembly into a bundle of longitudinally-aligned astrocytes with bipolar or multipolar processes, (v) is capable of forming a bundle of astrocytes; and/or (vi) has a morphology of an astrocyte. In some embodiments, an astrocyte is capable of self-assembly into a bundle of longitudinally aligned astrocytes with bidirectional (e.g., bipolar) or multidirectional processes. In some embodiments, an astrocyte has a “star-like” morphology, e.g., as observed in protoplasmic astrocytes. In some embodiments, an astrocyte has a linear morphology, e.g., as observed in fibrous astrocytes.
In some embodiments, an astrocyte has any one of characteristics (i) to (vi). In some embodiments, an astrocyte has any two of characteristics (i) to (vi). In some embodiments, an astrocyte has any three of characteristics (i) to (vi). In some embodiments, an astrocyte has any four of characteristics (i) to (vi). In some embodiments, an astrocyte has any five of characteristics (i) to (vi). In some embodiments, an astrocyte has all of characteristics (i) to (vi).
In some embodiments, an astrocyte is a cell that expresses GFAP, Ezrin, Robo2, S-100-beta, glutamine synthetase (GS) and/or Glutamate Aspartate Transporter (GLAST), or a combination thereof. In some embodiments, an astrocyte is a cell that expresses GFAP. In some embodiments, an astrocyte is a cell that expresses Ezrin. In some embodiments, an astrocyte is a cell that expresses Robo2. In some embodiments, an astrocyte is a cell that expresses S-100-beta. In some embodiments, an astrocyte is a cell that expresses glutamine synthetase (GS). In some embodiments, an astrocyte is a cell that expresses Glutamate Aspartate Transporter (GLAST). In some embodiments, an astrocyte is a cell that expresses GFAP, Ezrin, Robo2, S-100-beta, glutamine synthetase (GS) and Glutamate Aspartate Transporter (GLAST),
In some embodiments, an astrocyte is obtained, e.g., derived, from a stem cell, e.g., a GDMSC.
In some embodiments, an astrocyte is obtained from a central nervous system of an organism.
In some embodiments, an astrocyte is obtained using a method disclosed herein.
Disclosed herein, inter alia, are systems comprising: a biocompatible construct comprising a matrix; and a plurality of astrocytes, e.g., obtained from a GDMSC. Systems disclosed herein can be manufactured, e.g., assembled, in vitro or ex vivo. Systems disclosed herein can be implanted in a subject and can be used to induce regeneration, proliferation, differentiation and/or migration of a cell, e.g., a progenitor cell, a neuronal precursor cell, a neuroblast, a neuron, a glial cell, an astrocyte, and/or an endothelial cell. Systems disclosed herein can also be used to treat and/or ameliorate one or more symptoms of a neurodegenerative disorder or a neurological disorder in a subject. In some embodiments, a system disclosed herein is referred to as a Tissue Engineered Rostral Migratory Stream (TE-RMS).
In some embodiments, a system disclosed herein (e.g., a TE-RMS) comprises a plurality of astrocytes obtained according to a method disclosed herein. In some embodiments, a plurality of astrocytes in a system disclosed herein has one or more characteristics of an astrocyte, e.g., as disclosed herein.
In some embodiments, at least a portion of a plurality of astrocytes in a system disclosed herein aggregate, e.g., form a bundle, e.g., a bundle of astrocytes. In some embodiments, a substantial portion of a plurality of astrocytes in a system disclosed herein aggregate, e.g., form a bundle, e.g., a bundle of astrocytes. In some embodiments, a plurality of astrocytes in a system disclosed herein aggregate, e.g., form a bundle, e.g., a bundle of astrocytes.
In some embodiments, a plurality of astrocytes in a system disclosed herein comprise a bidirectional (e.g., bipolar) or multidirectional morphology.
In some embodiments, a system disclosed herein (e.g., a TE-RMS) further comprises one or more additional cells and/or one or more additional components. In some embodiments, an additional cell or component is introduced into a system: (i) concurrently with, before or after seeding of the plurality of astrocytes; (ii) during formation of an astrocyte bundle by the plurality of astrocytes; and/or (ii) after formation of an astrocyte bundle by the plurality of astrocytes.
In some embodiments, one or more additional components comprises a Robo-Slit entity. In some embodiments, a Slit-Robo entity is or comprises an agent that promotes activation and/or signaling from a Slit-Robo pathway
In some embodiments, one or more additional cells or components induces vascularization.
In some embodiments, a system disclosed herein further comprises one or more additional cells. In some embodiments, an additional cell is a cell other than an astrocyte or astrocytic cell. In some embodiment, an additional cell in a system disclosed herein induces vascularization. In some embodiments, an additional cell is an endothelial cell.
Also disclosed herein is a method of manufacturing a system disclosed herein, e.g., a TE-RMS. In some embodiments, a method of manufacturing a system disclosed herein, e.g., a TE-RMS, comprises (a) providing a biocompatible construct comprising a matrix and having a first end, a second end and a body; and (b) associating a biocompatible construct with a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC).
In some embodiments, associating a biocompatible construct with a plurality of astrocytes comprises seeding a plurality of astrocytes in a biocompatible construct. In some embodiments, a plurality of astrocytes is seeded on an inner surface of a biocompatible construct.
In some embodiments, a plurality of astrocyte is seeded at least once in a biocompatible construct. In some embodiments, a plurality of astrocyte is seeded twice in a biocompatible construct.
In some embodiments, a plurality of astrocytes is seeded at a cell density of about 0.1 million cells/ml to about 10 million cells/ml. In some embodiments, a plurality of astrocytes is seeded at a cell density of about 0.1 million cells/ml, about 0.2 million cells/ml, about 0.5 million cells/ml, about 1 million cells/ml, about 2 million cells/ml, about 3 million cells/ml, about 4 million cells/ml, about 5 million cells/ml, about 6 million cells/ml, about 7 million cells/ml, about 8 million cells/ml, about 9 million cells/ml, or about 10 million cells/ml. In some embodiments, a plurality of astrocytes is seeded at a cell density of about 1 million cells/ml.
In some embodiments, a plurality of astrocytes, e.g., in a system disclosed herein, comprises at least 500 cells, at least 1000 cells, at least 5000 cells, at least 10,000 cells, at least 15,000 cells, at least 20,000 cells, at least 40,000 cells, at least 80,000 cells, at least 100,000 cells, or at least 500,000 cells.
In some embodiments, a method of manufacturing a system disclosed herein comprises maintaining a system under conditions that promotes growth of at least one astrocyte in the plurality of astrocytes. In some embodiments, a method of manufacturing a system disclosed herein comprises maintaining a system under conditions that maintain viability of at least one astrocyte in the plurality of astrocytes.
In some embodiments, a method of manufacturing a system disclosed herein comprises forming an aggregate, e.g., a bundle of astrocytes, of at least a portion of the plurality of astrocytes.
Additional methods of manufacturing a system disclosed herein, e.g., a TE-RMS, are disclosed in International Application PCT/US2015/065353 filed on Dec. 11, 2015, the entire contents of which are hereby incorporated by reference.
In some embodiments, disclosed herein is a system comprising a plurality of astrocytes wherein at least a portion of said astrocytes comprises one or more aligned and/or elongated astrocyte processes. In some embodiments, an astrocyte process has a length of at least 0.2 mm, at least 0.5 mm, at least 1 mm, at least 2 mm, at least 5 mm or at least 10 mm. Also disclosed herein is a method of manufacturing a system comprising a plurality of astrocytes wherein at least a portion of said astrocytes comprise one or more aligned and/or elongated astrocyte processes.
In some embodiments, a system disclosed herein is a three-dimensional system.
A biocompatible construct disclosed herein comprises a matrix, e.g., as disclosed herein, and a first end, a second end, and a body. In some embodiments, a matrix of a biocompatible construct comprises an inner surface and/or an outer surface. In some embodiments, an inner surface of a biocompatible construct defines a luminal core.
In some embodiments, a matrix of a biocompatible construct comprises a scaffold. In some embodiments, a matrix of a biocompatible construct comprises a component described herein, e.g., a component of an inner surface as described herein, and/or a component of an outer surface as described herein. In some embodiments, a matrix of a biocompatible construct comprises one or more additional components, e.g., as disclosed herein.
As an example, an outer surface of a biocompatible construct disclosed herein comprises at least one hydrogel (e.g., as described herein). In some embodiments, a hydrogel comprises a hydrophilic biopolymer and/or a synthetic polymer. In some embodiments, a hydrogel is at least partially cross-linked, wherein the cross-linking optionally increases stiffness, reduces porosity, and/or increases degradation time. In some embodiments, a hydrophilic biopolymer comprises one or more of agarose, hyaluronic acid, chitosan, alginate, collagen, dextran, pectin, carrageenan, polylysine, gelatin, hyaluronic acid, fibrin, and methylcellulose.
In some embodiments, a hydrophilic biopolymer comprises agarose. In some embodiments, a hydrophilic biopolymer comprises hyaluronic acid. In some embodiments, a hydrophilic biopolymer comprises chitosan. In some embodiments, a hydrophilic biopolymer comprises alginate. In some embodiments, a hydrophilic biopolymer comprises collagen. In some embodiments, a hydrophilic biopolymer comprises dextran. In some embodiments, a hydrophilic biopolymer comprises pectin. In some embodiments, a hydrophilic biopolymer comprises carrageenan. In some embodiments, a hydrophilic biopolymer comprises polylysine. In some embodiments, a hydrophilic biopolymer comprises gelatin. In some embodiments, a hydrophilic biopolymer comprises hyaluronic acid. In some embodiments, a hydrophilic biopolymer comprises fibrin. In some embodiments, a hydrophilic biopolymer comprises methylcellulose.
In some embodiments, a hydrophilic biopolymer comprises hyaluronic acid. In some embodiments, wherein a hyaluronic acid is or comprises methacrylated HA (MeHA).
In some embodiments, a hydrophilic biopolymer comprises agarose. In some embodiments, an agarose is at about 0.25-30%, about 0.25%-3%, about 0.5%-3%, about 1-20%, about 1.5-10%, about 2-9%, about 2.5-8%, or about 3-7%. In some embodiments, the agarose is at about 0.25-29%, about 0.25-28%, 0.25-%, about 0.25-27%, about 0.25-26%, about 0.25-25%, about 0.25-24%, about 0.25-23%, about 0.25-22%, about 0.25-21%, about 0.25-20%, about 0.25-19%, about 0.25-18%, about 0.25-17%, about 0.25-16%, about 0.25-15%, about 0.25-14%, about 0.25-13%, about 0.25-12%, about 0.25-11%, about 0.25-10%, about 0.25-9%, about 0.25-8%, about 0.25-7%, about 0.25-6%, about 0.25-5%, about 0.25-4%, about 0.25-3%, about 0.25-2%, about 0.25-1%, about 0.25-0.5%, about 0.5-30%, about 1-30%, about 2-30%, about 3-30%, about 4-30%, about 5-30%, about 6-30%, about 7-30%, about 8-30%, about 9-30%, about 10-30%, about 11-30%, about 12-30%, about 13-30%, about 14-30%, about 15-30%, about 16-30%, about 17-30%, about 18-30%, about 19-30%, about 20-30%, about 21-30%, about 22-30%, about 23-30%, about 24-30%, about 25-30%, about 26-30%, about 27-30%, about 28-30%, about 29-30%.
In some embodiments, an agarose is at about 0.25%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%. In some embodiments, an agarose is at about 3%.
An inner surface of a biocompatible construct disclosed herein, in some embodiments, comprises one or more extracellular matrix (ECM) components. In some embodiments, an ECM component comprises collagen, laminin, fibronectin, hyaluronic acid, or a combination thereof. In some embodiments, an ECM component comprises collagen. In some embodiments, an ECM component comprises laminin. In some embodiments, an ECM component comprises fibronectin. In some embodiments, an ECM component comprises hyaluronic acid.
In some embodiments, an ECM component comprises collagen. In some embodiments, a collagen is at a concentration of about 0.1-10 mg/ml. In some embodiments, a collagen is at a concentration of about 0.1-9 mg/ml, about 0.1-8 mg/ml, about 0.1-7 mg/ml, about 0.1-6 mg/ml, about 0.1-5 mg/ml, about 0.1-4 mg/ml, about 0.1-3 mg/ml, about 0.1-2 mg/ml, about 0.1-1 mg/ml, about 0.1-0.9 mg/ml, about 0.1-0.8 mg/ml, about 0.1-0.7 mg/ml, about 0.1-0.5 mg/ml, about 0.5-10 mg/ml, about 0.6-10 mg/ml, about 0.7-10 mg/ml, about 0.8-10 mg/ml, about 0.9-10 mg/ml, about 1-10 mg/ml, about 2-10 mg/ml, about 3-10 mg/ml, about 4-10 mg/ml, about 5-10 mg/ml, about 6-10 mg/ml, about 7-10 mg/ml, about 8-10 mg/ml, about 9-10 mg/ml. In some embodiments, a collagen is at a concentration of about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml. In some embodiments, a collagen is at a concentration of about 1 mg/ml.
In some embodiments, an ECM component comprises laminin. In some embodiments, a laminin is at a concentration of about 0.1-9 mg/ml, about 0.1-8 mg/ml, about 0.1-7 mg/ml, about 0.1-6 mg/ml, about 0.1-5 mg/ml, about 0.1-4 mg/ml, about 0.1-3 mg/ml, about 0.1-2 mg/ml, about 0.1-1 mg/ml, about 0.1-0.9 mg/ml, about 0.1-0.8 mg/ml, about 0.1-0.7 mg/ml, about 0.1-0.5 mg/ml, about 0.5-10 mg/ml, about 0.6-10 mg/ml, about 0.7-10 mg/ml, about 0.8-10 mg/ml, about 0.9-10 mg/ml, about 1-10 mg/ml, about 2-10 mg/ml, about 3-10 mg/ml, about 4-10 mg/ml, about 5-10 mg/ml, about 6-10 mg/ml, about 7-10 mg/ml, about 8-10 mg/ml, about 9-10 mg/ml.
In some embodiments, a laminin is at a concentration of about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml, about 0.9 mg/ml, about 1 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml. In some embodiments, a laminin is at a concentration of about 1 mg/ml.
A biocompatible construct disclosed herein can have one or more characteristics described herein and/or can be part of a system disclosed herein. In some embodiments, a biocompatible construct disclosed herein is a linear construct.
In some embodiments, a system comprising a biocompatible construct disclosed herein has an outer diameter of about 0.07-70 mm, about 0.1-70 mm, 0.2-70 mm, about 0.3-70 mm, about 0.4-70 mm, about 0.5-70 mm, about 0.6-70 mm, about 0.7-70 mm, about 0.07-60 mm, about 0.07-50 mm, about 0.07-40 mm, about 0.07-30 mm, about 0.07-20 mm, about 0.07-10 mm, about 0.07-5 mm, about 0.07-1 mm, about 0.07-0.5 mm, about 0.07-0.7 mm, about 0.1-1 mm, about 0.2-5 mm, about 0.3-1 mm, about 0.4-0.9 mm, or about 0.5-0.8 mm. In some embodiments, a system comprising a biocompatible construct disclosed herein has an outer diameter of about 0.07 mm, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 1 mm, about 5 mm, about 10 mm, about 20 mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, or about 70 mm. In some embodiments, a system comprising a biocompatible construct disclosed herein has an outer diameter of about 0.7 mm.
In some embodiments, a system comprising a biocompatible construct disclosed herein has an inner diameter of about 0.03-30 mm, about 0.05-30 mm, about 0.1-30 mm, about 0.15-30 mm, about 0.2-30 mm, about 0.25-30 mm, about 0.3-30 mm, about 0.03-25 mm, about 0.03-20 mm, about 0.03-15 mm, about 0.03-10 mm, about 0.03-5 mm, about 0.03-1 mm, about 0.03-0.5 mm, or about 0.03-0.3 mm. In some embodiments, a system comprising a biocompatible construct disclosed herein has an inner diameter of about 0.03 mm, about 0.05 mm, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.5 mm, about 1 mm, about 5 mm, about 10 mm, about 15 mm, about 20 mm, about 25 mm or about 30 mm. In some embodiments, a system comprising a biocompatible construct disclosed herein has an inner diameter of about 0.3 mm.
A system disclosed herein comprising a plurality of astrocytes obtained from a GDMSC can be used, e.g., as an in vitro test bed; for neural tissue engineering approaches; to promote differentiation, proliferation and/or migration of cells; and/or to treat or ameliorate a symptom of one or more neuropathologies (e.g., as described herein).
In some embodiments, a system disclosed herein is used in a method of promoting cell migration, differentiation and/or proliferation, comprising providing a system comprising: (a) a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell; and (b) a biocompatible construct comprising a first end, a second end and a body comprising a luminal core.
In some embodiments, a method promotes migration, differentiation and/or proliferation of a cell endogenous to a subject in which a biocompatible construct is implanted. In some embodiments, an endogenous cell comprises a neural precursor cell, a neuroblast, a neuron, a progenitor cell, a glial cell, an astrocyte, and/or an endothelial cell.
In some embodiments, a method promotes migration of one or more cells provided in a system; or one or more cells derived from, differentiated from or a progenitor of a cell provided in a system.
In some embodiments, migration of one or more cells occurs within a system, throughout a system, out of a system or into a system.
In some embodiments, a system disclosed herein is used in a method of treating and/or ameliorating one or more symptoms of a neurodegenerative disorder or a neurological disorder in a subject, comprising providing to the subject a system comprising: (a) a plurality of astrocytes derived from at least one gingiva-derived mesenchymal stem cell (GDMSC); and (b) a biocompatible construct comprising a matrix and having a first end, a second end and a body.
In some embodiments, a neurodegenerative disorder comprises a disorder with injury or degeneration to one or more neurons.
In some embodiments, a neurodegenerative disorder comprises brain injury or spinal cord injury.
In some embodiments, brain injury comprises acute brain injury, degenerative brain injury, traumatic brain injury (TBI), or chronic brain injury.
In some embodiments, a neurological disorder comprises: Parkinson's, Alzheimer's, Huntington's, prion disease, motor neuron disease, spinocerebellar ataxia, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), encephalitis, epilepsy, head and brain malformations, or hydrocephalus.
In some embodiments, a subject is a human.
In some embodiments, of any of the methods disclosed herein, a system is implanted into a subject. In some embodiments, a system is implanted into the brain of a subject. In some embodiments, a system is implanted: (i) at, near or within a brain lesion in a subject; (ii) at, near or within an area in the brain with insufficient neurons or neuronal connections, or with damaged neurons or neuronal connections; (iii) at, near or within a subventricular zone, an endogenous rostral migratory stream or a neurogenic niche; and/or (iv) at, near or within a region of a brain affected by a brain injury, a neurodegenerative disease or disorder, or a neurodevelopmental disease or disorder.
In some embodiments, a system is characterized in that when implanted into an organism it ameliorates or reduces severity of one or more symptoms of a disorder.
In some embodiments, a system is characterized in that when implanted into an organism it promotes migration of one or more cells.
In some embodiments, migration of one or more cells is compared to a comparator. In some embodiments, a comparator comprises an organism implanted with an otherwise similar system without a plurality of astrocytes; or without a plurality of astrocytes derived from at least one GDMSC.
In some embodiments, one or more migrating cells comprises one or more endogenous host cells. In some embodiments, an endogenous host cell comprises a neural precursor cell, a neuroblast, a neuron, a progenitor cell, a glial cell, an astrocyte, and/or an endothelial cell.
In some embodiments, one or more migrating cells comprises: one or more cells provided in a system; or one or more cells derived from, differentiated from or a progenitor of a cell provided in a system. In some embodiments, migration of one or more cells occurs within a system, throughout a system, out of a system or into a system.
In some embodiments, migration of one or more cells occurs to a site of implantation, or away from a site of implantation.
In some embodiments, a method disclosed herein is characterized in that when implanted in an organism, it promotes differentiation of one or more cells, e.g., an endogenous cell in which a system is implanted, or a cell provided in or derived from a system disclosed herein.
In some embodiments, a method disclosed herein is characterized in that when implanted in an organism, it promotes proliferation of one or more cells, e.g., an endogenous cell in which a system is implanted, or a cell provided in or derived from a system disclosed herein.
The rostral migratory stream (RMS) facilitates neuroblast migration from the subventricular zone to the olfactory bulb throughout adulthood. Brain lesions attract neuroblast migration out of the RMS, but resultant regeneration is insufficient. Increasing neuroblast migration into lesions has improved recovery in rodent studies. Techniques for fabricating an astrocyte-based Tissue-Engineered RMS (TE-RMS) intended to redirect endogenous neuroblasts into distal brain lesions for sustained neuronal replacement were previously developed. This Example demonstrates that astrocytes can be derived from adult human gingiva mesenchymal stem cells and used for TE-RMS fabrication. This Example also shows that key proteins enriched in the RMS are enriched in TE-RMSs. Furthermore, the human TE-RMS facilitated directed migration of immature neurons in vitro. Finally, human TE-RMSs implanted in athymic rat brains redirect migration of neuroblasts out of the endogenous RMS. By emulating the brain's most efficient means for directing neuroblast migration, in some embodiments, a TE-RMS offers a promising new approach to neuroregenerative medicine.
Adult neurogenesis continues in the mammalian brain in the subgranular zone of the dentate gyrus and the subventricular zone (SVZ) surrounding the lateral ventricles1,2. Neural precursor cells (NPCs) in the SVZ can differentiate into neuroblasts and migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB) where they mature into interneurons and integrate into existing circuitry3-6. Neuroblasts migrate in chain formation at a rate between 30-70 μm/hour (0.72-1.68 mm/day)7-10 along astrocytes that comprise the RMS (
Endogenous neurogenesis is upregulated in the SVZ following brain injury16-18. Increased neurogenesis has been reported in the rodent SVZ following multiple experimental models of acquired brain injury, including but not limited to stroke19-28, controlled cortical impact brain injury29-31, and lateral fluid percussion brain injury32,33. Following brain injury, these newly formed NPCs can mature into neuroblasts, divert from the SVZ/RMS, and migrate toward injured brain regions27,28,34,35 (
Neural tissue engineering has introduced the possibility of developing customized therapies to enhance neuronal regeneration following traumatic brain injury. A variety of biomaterial and tissue engineering technologies have been developed to enhance the neurogenic potential of the SVZ and redirect the migration of SVZ neuroblasts to neuron-deficient brain regions following various experimental brain injuries (for recent review, see Purvis et al., 202049). The evidence from tissue engineering techniques, along with that of pharmacological and genetic approaches, collectively demonstrates that experimental intervention to enhance the brain's intrinsic repair mechanism to replace lost or damaged neurons with endogenous SVZ NPCs can improve recovery after acquired brain injury. However, while promising, these interventions have thus far only afforded transient re-direction of neuroblasts, while in some embodiments, a sustained influx of new neurons is likely required for functional improvements across a spectrum of brain injury severities.
Three-dimensional tissue engineered “living scaffolds” that replicate specific neuroanatomical features of neural architecture and/or circuitry have been previously developed. Implantation of these fully formed, living microtissue scaffolds in vivo has allowed successful facilitation of nervous system repair by replacing and/or augmenting lost circuitry50-54 and facilitating axonal regeneration and pathfinding55. In addition, the first Tissue-Engineered Rostral Migratory Stream (TE-RMS) was recently developed, which is an implantable scaffold designed to replicate the endogenous RMS56-58. This engineered neuronal replacement strategy replicates the only known mechanism for continual, long-distance neuroblast redirection that occurs intrinsically within the adult brain via the RMS. By recapitulating the structure and function of the glial tube at the core of the RMS, in some embodiment, a TE-RMS is designed to promote sustained delivery of neuroblasts to neuron-deficient regions following injury or neurodegenerative disease. In some embodiments, it is expected that stable, long-term neuroblast redirection via this engineered living scaffold will set this technology apart from previous strategies that have typically induced transient neuronal redirection.
The TE-RMS is biofabricated within a small-diameter agarose microcolumn that promotes astrocyte bundling with extracellular matrix and self-assembly into long, longitudinally-aligned cables (
In the current study, protein expression in astrocytes of the TE-RMS was compared to that of the glial tube in the endogenous RMS. This Example also reports the ability to fabricate the TE-RMS from a readily available source of adult human gingiva mesenchymal stem cells (GMSCs) from which astrocytes can be derived within one week using non-genetic techniques without the need for dedifferentiation. This enhances the translational potential of this technology by introducing the possibility that, in some embodiments, with further development, human autologous TE-RMS implants can be created. The data herein also demonstrates that the human TE-RMS facilitates immature neuronal migration in vitro. Finally, it is reported that implantation of the human TE-RMS into the athymic rat brain facilitates migration of endogenous neuroblasts out of the native RMS and throughout the TE-RMS, providing surgical feasibility and proof-of-concept evidence for this technology.
Previous studies have characterized the enrichment of protein markers in the glial tube astrocytes of the RMS as compared to surrounding protoplasmic astrocytes. As such, the expression and distribution of these enriched proteins was compared in astrocytes of the native RMS versus astrocytes of the TE-RMS. Fluorescence immunohistochemistry (IHC) was applied in sagittally-sectioned FFPE adult rat brains (n=5 brains) to label GFAP, Ezrin, and Robo2 (
The development of fabrication techniques for the TE-RMS were previously reported, in which optimal microcolumn diameter, collagen concentration, media constituents, seeding density, and other factors were evaluated to facilitate astrocyte self-assembly into longitudinally-aligned, tightly bundled cords over a period of just 8 hours. Data on the similarities in the morphology and structural arrangement of rat TE-RMS astrocytes compared with astrocytes of the endogenous RMS were also previously disclosed56-58. In this Example, the hypothesis that TE-RMS astrocytes also recapitulate the enhanced expression of GFAP, Ezrin, and Robo2 observed in the endogenous RMS was evaluated (as verified in the experiments of
Astrocytes can be Derived from Adult Human GMSCs and Used for TE-RMS Fabrication
To identify a clinically relevant starting biomass for TE-RMS fabrication, the efficacy of a novel differentiation protocol to derive astrocytes from GMSCs was evaluated. This protocol was adapted from a previously published method for astrocyte derivation from oral mucosal stem cells59. Here, this non-genetic derivation protocol was successful applied to GMSCs from three deidentified adult human patients obtained via minimally invasive punch biopsy. After the derivation process—which takes less than a week—the cultured cells from each subject expressed astrocytic proteins Glutamine Synthetase (GS), Glutamate Aspartate Transporter (GLAST), GFAP, and S100-β and were negative for the endothelial marker CD31 (
Fluorescence ICC with laser confocal microscopy was used to confirm that TE-RMSs fabricated from human GMSC-derived astrocytes expressed GFAP, Ezrin, and Robo2 (
Looking beyond replicating the morphology, arrangement, and protein expression of the endogenous RMS, in some embodiments, a human TE-RMS is expected to have a substantially similar function as an endogenous RMS to be used for clinical application or as an in vitro test bed. Techniques from a previously reported migration assay in the rat TE-RMS were adapted58, and migration of immature rat cortical neurons through the human TE-RMS was assessed as compared to acellular collagen-coated or collagen+laminin-coated control columns (
The Human TE-RMS Redirects Migration of Neuroblasts from the Rat RMS In Vivo
Finally, in vivo experiments with stereotactic implantation of human TE-RMSs into the brains of athymic rats were performed to test surgical feasibility and proof-of-principle for redirecting neuroblast migration away from the endogenous RMS (
The TE-RMS is the first biomimetic implantable microtissue designed to redirect the migration of endogenous neuroblasts out of the RMS and into distal lesions, intended to, in some embodiments, provide sustained delivery to replace lost neurons and improve functional recovery. In pursuit of this objective, the TE-RMS was developed to emulate the brain's only existing method for transporting neuroblasts to a distal area for neuronal replacement. Whereas prior studies have transplanted exogenous fetal grafts, single cell suspensions, or cells in 3-D matrices, the method disclosed herein is considerably different in that the living cytoarchitecture of the TE-RMS—mimicking the architecture the RMS—is fully biofabricated in vitro and then precisely delivered to unlock the regenerative potential of the brain's own endogenous neuroblasts. In the current study it was confirmed that the TE-RMS is enriched in Ezrin and Robo2, both of which are similarly enriched in the endogenous RMS and important for facilitating neuroblast migration. This Example also reports a new method for deriving astrocytes from adult human GMSCs adapted from a previous protocol applied to oral mucosa, potentially providing a minimally invasive autologous starting biomass for patients. Indeed, utilizing these cells for fabrication produces a human TE-RMS consisting of tightly-bundled, bidirectional, longitudinally-aligned, astrocytes enriched in GFAP, Ezrin, and Robo2. Furthermore, the human TE-RMS facilitated in vitro migration of immature neurons at rates within the range observed for migration of neuroblasts in the endogenous RMS. Finally, the data disclosed herein provides in vivo evidence of surgical feasibility for human TE-RMS implantation into athymic rat brains spanning from the RMS to the cortex, and proof-of-principle evidence that the human TE-RMS can redirect migration of endogenous rat neuroblasts out of the RMS and into cortex with no overt negative histological or behavioral effects on test subjects.
While the direct implantation of neural stem cells into injury sites has been shown to improve outcome by providing neurotrophic factors, and recent work has demonstrated that integration of human induced pluripotent stem cell (iPSC) derived neurons in rat cortex is possible62, appropriate maturation and integration of these exogenous cells to functionally replace lost neurons remains a significant challenge63-65. Furthermore, each delivery of exogenous neural stem cells requires invasive surgery, therefore these proposed treatment strategies typically involve only a single bolus delivery of exogenous stem cells. In contrast, a single surgical procedure to implant a TE-RMS could, in some embodiments, provide sustained delivery of endogenous NPCs by emulating the brain's own strategy for relocating and integrating new neurons. In addition, strategies for direct implantation of exogenous stem cells often rely on iPSCs that must be dedifferentiated from a more mature cell source, and therefore carry risks related to the retention of epigenetic memory from the original cell source (i.e. leading to de-differentiation)66. In contrast, a TE-RMS provides the brain's own endogenous NPCs that do not require any prior de-differentiation, and therefore in some embodiments do not suffer from the phenotypic abnormalities sometimes associated with epigenetic memory in iPSCs. Genetic modification of neuroblasts may not represent a translational therapeutic strategy, but it does support the feasibility of enhancing neuroblast migration into lesions to facilitate neuroregeneration. A TE-RMS disclosed herein, in some embodiments, can be precisely implanted to span SVZ to lesion, providing a migratory pathway to augment and amplify this natural regenerative response of endogenous SVZ neuroblasts after injury without the need for genetic manipulation.
Increasing delivery of neuroblasts into lesions after injury has also been approached through pharmacological strategies and implantation of acellular permissive substrates49,67. Pharmacological approaches have focused mainly on administration of neurotrophic factors. Intraventricular infusion of various combinations of neurotrophic factors including epidermal growth factor, erythropoietin, fibroblast growth factor, vascular endothelial growth factor, and others has been shown to improve short-term functional recovery by enhancing proliferation in the SVZ after injury, in turn increasing the number of neuroblasts migrating into lesions by virtue of increasing the overall number of neuroblasts39. There is extensive evidence in humans and animal models for exercise-induced improvements in functional recovery after brain injury, and the effects are largely attributed to increased neuronal plasticity and proliferation of endogenous NPCs in response to exercise-induced increases in brain-derived neurotrophic factor68,69. In the current study, in vitro chain migration of immature cortical neurons through a TE-RMS was observed, whereas columns loaded with ECM only (1 mg/mL collagen+1 mg/mL laminin) promoted neurite outgrowth of these immature cells and did not promote migratory behavior. The data disclosed herein is distinct from previous research demonstrating neuroblast migration along planar laminin and collagen in 2D culture in vitro (73). This difference in cell behavior is likely due to difference in immature neuronal phenotype (SVZ-derived versus cortical) as well as ECM preparation across in vitro studies leading to differential binding of immature neurons to laminin. Indeed, migration and maturation of neuroblasts along the RMS relies on complex, dynamic signaling between astrocytes and neuroblasts74-76 Unlike extracellular-matrix-based constructs that offer a permissive, acellular substrate70,72,73, a TE-RMS disclosed herein possesses a unique, living astrocytic microtissue makeup that can provide directional, structural, and/or neurotrophic support, making it capable, e.g., of sending as well as responding to complex signals with migrating neuroblasts and the local micro-environment. Relying on an acellular substrate infused with a handful of signaling molecules is akin to trying to coordinate a complex project in a foreign language of which you speak only a few words and cannot hear or respond to anything anyone else is saying, versus the living microtissue TE-RMS that provides total fluency in the language and engagement in dynamic, collaborative conversations.
Glial tube astrocytes possess several features that make them unique among the widely heterogeneous astrocyte milieu. They possess a bidirectional morphology, extending processes in opposite directions along the glial tube in parallel with each other to form a cord-like bundle. It has been previously established that these structural features are recapitulated in the TE-RMS56-58. Glial tube astrocytes are also enriched in several proteins important for facilitating neuroblast migration. Ezrin—a member of the cytoskeleton-membrane linking ERM (Ezrin, radixin, moesin) protein family—is enriched in the astrocytes of the glial tubes, while its cousin radixin is enriched in migrating neuroblasts15,77. In the endogenous RMS, astrocytic Robo2 receptors detect Slit1 protein released by neuroblasts, and this signaling results in tunnel formation in the astrocytic meshwork of the glial tubes to facilitate neuroblast migration78. In this study, previous reports of enrichment of GFAP, Ezrin, and Robo2 in the glial tube astrocytes of the endogenous rat RMS were evaluated. Using the same methods, previous morphological and structural analyses were expanded to show that the TE-RMS—fabricated from either primary rat astrocytes or human GMSC-derived astrocytes—is also enriched in GFAP, Ezrin, and Robo2.
Building on this concept, in some embodiments, mimicking the RMS may also facilitate maturation of neuroblasts during migration along the TE-RMS, allowing the new neurons to functionally replace lost neurons after degeneration has occurred instead of merely acting as neurotrophic “factories” in the acute and sub-acute time periods. This strategy to enable replacement of lost neurons well after acquired brain injury or neurodegeneration is highly unique, and presents an innovative approach to repairing currently untreatable injuries affecting millions of patients. In some embodiments, their miniature form factor allows for minimally invasive, stereotactic delivery into the brain. In rat models, the ability to precisely microinject similar allogeneic neuronal microtissue constructs, which maintain their pre-transplant architecture, survive, and integrate into the native nervous system was previously disclosed52-54,79,80, and TE-RMS implant experiments included in this study further demonstrate the surgical feasibility of this strategy.
In this study, the successful derivation of astrocytes from adult human GMSCs, their suitability as a starting biomass for TE-RMS fabrication, and proof-of-concept evidence that these human TE-RMSs facilitate neuroblast migration in vitro and in vivo is reported. Like iPSCs, the GMSCs offer minimally-invasive access to a patient-specific autologous starting biomass. However, the one-week GMSC-to-astrocyte derivation process takes only a fraction of the time of iPSC derivations, due in part to the fact that dedifferentiation is unnecessary in the case of GMSCs. That lack of a dedifferentiation step also means that GMSCs do not carry risks associated with epigenetic memory66. The appeal of a patient-specific starting biomass is primarily due to avoiding the dangers of immune rejection.
In some embodiments, a TE-RMS strategy is intended, e.g., for neuronal replacement and functional regeneration. In some embodiments, a TE-RMS disclosed herein can be implanted after the acute period to allow the so-called “hostile” environment—marked by dysregulated interstitial tissue, ongoing cell death, and widespread inflammation—to dissipate. In some embodiments, this strategy is also intended to serve as a means for gradual, sustained delivery of neuroblasts into injury sites. In some embodiments, the potential spectrum of cell fates when redirecting neuroblasts to various discrete brain areas can be determined. In some embodiments, a TE-RMS can be used as a biofidelic test bed for performing efficient and precise mechanistic studies. A human TE-RMS disclosed herein has opened new avenues for potential study, and a promising novel approach to leveraging the endogenous regenerative potential of the brain.
All procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. For TE-RMS fabrication, primary astrocytes were harvested from the cortices of postnatal day 0-1 Sprague-Dawley rat pups (Charles River, Wilmington, MA). Cells were dissociated57 and cultured in DMEM/F12 medium supplemented with 10% FBS and 1% Penicillin-Streptomycin in a 37° C./5% CO2 cell culture incubator. Over several weeks, astrocyte cultures were maintained in tissue culture flasks and passaged at 80% confluency to purify the astrocyte population. Astrocytes between passages 4-10 were utilized for all in vitro experiments. For in vitro migration of immature neurons through the TE-RMS, primary rat neurons were dissociated from cortices of embryonic day 18 (E18) rats57. Following tissue dissociation with trypsin-EDTA and DNAse I, a cell solution with a density of 1.0-2.0×106 cells/mL was prepared. 12 μL of this solution was transferred to each well in the pyramidal micro-well array. The plate containing these micro-wells was centrifuged to produce cell aggregates.
Hydrogel micro-columns composed of 3% agarose were utilized to induce the alignment of astrocytes and create the TE-RMS57. Agarose was dissolved and heated in Dulbecco's phosphate-buffered saline (DPBS). An acupuncture needle (diameter=300 μm) was inserted into the bottom opening of a bulb dispenser. A glass capillary tube (inner diameter=701 μm) was inserted over the needle external to the bulb and secured to the rubber section of the bulb dispenser. Warm agarose was drawn into capillary tube with the needle in the center. After allowing the agarose to cool, the capillary tube was carefully removed, and the micro-columns were gently pushed off the needle into DPBS and sterilized by UV light for 30 minutes. Micro-columns had an outer diameter of 701 μm and an inner diameter of 300 μm. Optimal micro-column dimensions for inducing alignment and bundling of astrocytes were determined based on previous experiments56.
Hydrogel micro-columns were cut with angled forceps to a length of 4 mm. The inner lumen of the micro-columns was loaded with 1 mg/mL rat tail type 1 collagen diluted in cell culture medium. The collagen-loaded constructs were incubated for 3 hours at 37° C./5% CO2 to allow for collagen polymerization and dehydration yielding in a hollow microcolumn with the surface of the inner lumen coated in collagen. After the complete collagen polymerization, the inner lumen of the micro-columns was seeded with astrocytes in serum-free co-culture media at a density of ˜1 million cells/mL (optimal seeding density confirmed by Winter et al., 201656). Co-culture media consisting of Neurobasal medium supplemented with 2% B-27, 1% G-5, 0.25% L-Glutamine, and 1% Penicillin-Streptomycin induced the astrocytes into a process-bearing phenotype. Columns were seeded twice with astrocytes to ensure that the entire interior of each micro-column was filled with cells. Following astrocyte seeding, columns were incubated at 37° C./5% CO2 for one hour and subsequently reinforced with 1 mg/mL collagen. Collagen reinforcement provided more ECM to the astrocytes, helping to prevent collapse during astrocyte bundling. Following reinforcement, astrocyte-loaded columns were incubated for another hour at 37° C./5% CO2, flooded with warm co-culture media, and returned to the cell culture incubator. Over a relatively short time period of ˜8 hours, the astrocytes extend processes to gather collagen and self-assemble into a bundled cord of longitudinally-aligned astrocytes with bidirectional processes, effectively forming TE-RMSs. For experimental purposes, TE-RMSs were utilized 24 hours after astrocyte seeding. Acellular collagen control columns for in vitro migration assays were prepared as above but with no addition of cells. For acellular collagen/laminin columns, a mixture of 1 mg/mL collagen and 1 mg/mL mouse laminin diluted in cell culture medium was loaded into micro-columns. Following extracellular matrix polymerization at 37° C./5% CO2 (approximately 3 hours), columns were flooded with warm co-culture media and returned to the cell culture incubator.
TE-RMS Extraction from Microcolumns for Immunocytochemistry
Following overnight bundling of astrocytes and formation of the TE-RMS, astrocytic bundles were extracted from hydrogel micro-columns onto glass coverslips using surgical forceps and a stereoscope for visual guidance. TE-RMSs were slowly drawn out of the micro-columns into a bead of collagen diluted in culture media and left to dry for 15 minutes at 37° C./5% CO2 to facilitate coverslip adhesion prior to fixation. Extraction was not performed with migration assay columns as it would disrupt the neuronal aggregate, and they were instead fixed, stained, and imaged within the microcolumns to keep the assay system intact.
Astrocyte Derivation from Adult Human GMSCs
Healthy human gingival tissues were obtained as remnants of discarded tissues under the approved Institutional Review Board (IRB) protocol at University of Pennsylvania. All procedures and methods were carried out in accordance with relevant guidelines and regulations. Informed consents were obtained from all participating human subjects for the collection of fresh tissues. Mesenchymal stem cells isolated from de-identified human gingiva were run through a non-genetic process similar to a method previously applied to derive astrocytes from human oral mucosal stem cells59. The derivation takes less than a week, and was accomplished in collaboration with the Le Laboratory at the University of Pennsylvania, with whom techniques for deriving neural crest stem cells and Schwann cell-like cells from GMSCs were previously developed82-84 The derivation began with 72 hour incubation in serum-free low-glucose DMEM with 100 μg/ml streptomycin, 100 U/ml penicillin, 1250 U/ml Nystatin, and 2 mM glutamine supplemented with 20 ng/ml N2 supplement (Thermo Scientific), basic fibroblast growth factor 2 (PeproTech, Rocky Hill, NJ), and epidermal growth factor (PeproTech). After 72 hours the media was replaced with serum-free low-glucose DMEM with 100 μg/ml streptomycin, 100 U/ml penicillin, 1250 U/ml Nystatin, and 2 mM glutamine supplemented with 1 mM dibutyryl cyclic AMP (Sigma-Aldrich), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 50 ng/ml Neuregulin (PeproTech), and 1 ng/ml platelet-derived growth factor (PeproTech). After derivation, astrocytes were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% Penicillin-Streptomycin and passaged at 80% confluency with mechanical perturbation prior to trypsinization to purify the astrocyte population. Cells between passages 4-10 were utilized for all in vitro and in vivo experiments.
Following overnight bundling of astrocytes and formation of human GMSC-derived TE-RMSs, immature rat cortical neuronal aggregates were placed into one end of the micro-columns using surgical forceps and a stereoscope for visual guidance. Aggregates were placed such that they contacted one end of the fully formed TE-RMS within the column and placed in the same relative position in the acellular collagen and collagen/laminin columns. Columns loaded with aggregates were returned to the 37° C./5% CO2 cell culture incubator and were fixed 72 hours following neuronal aggregate seeding.
Athymic (immunodeficient) adult male rats (Strain RNU 316 homozygous; Charles River, Wilmington, MA) were maintained under isoflurane anesthesia and mounted in a stereotaxic frame. Subjects were subcutaneously administered bupivacaine (2.0 mg/kg) for local analgesia. Bilateral craniotomies were performed +2.5 mm anterior to bregma and +1 mm from midline on either side. Immediately prior to implantation, a 4 mm human GMSC-derived TE-RMS or acellular collagen control microcolumn was drawn into a thin-walled 21XX-gauge (813 μm outer diameter, 737 μm inner diameter) needle in warm DPBS. The needle was centered over the craniotomy and lowered at a rate of 2 mm/min. The column was delivered into the brain, and the needle was slowly retracted at a rate of 1 mm/min while the plunger was fixed in place to deliver the column without expulsive force. Subjects (n=6) were implanted bilaterally with a human GMSC-derived TE-RMS construct in the right hemisphere and an acellular collagen control microcolumn in the left. Stereotaxic coordinates (AP+2.5; ML+1; DV−5 mm relative to bregma) provided consistent implantation of all columns spanning from the RMS to the cortex. Following column delivery, the craniotomies were covered with a thin sterile PDMS disc and bone wax and the scalp was sutured. Subjects were administered slow-release meloxicam (4.0 mg/kg) for sustained post-surgical analgesia.
The endogenous RMS was analyzed via immunohistochemistry in sagittal brain sections from 5 archival formalin-fixed paraffin-embedded (FFPE) adult Sprague Dawley rat brains. Brain blocks near midline were sliced into 8 μm sections and mounted on slides. Slides likely to contain RMS were deparaffinized, rehydrated, and underwent heat-induced epitope retrieval in TRIS-EDTA. Slides were then blocked in horse serum for 30 minutes. Primary antibodies were applied in 1× Optimax buffer overnight at 4° C. including mouse anti-Ezrin (1:50) (Sigma-Aldrich Cat #E8897, RRID: AB_476955), goat anti-glial fibrillary acidic protein (GFAP) (1:1000) (Abcam Cat #ab53554, RRID: AB_880202), and rabbit anti-Robo2 (1:50) (Novus Cat #NBP1-81399, RRID: AB_11013687). Slides were then rinsed in PBS/Tween and incubated in Alexa secondary antibodies (1:500) in 1× Optimax buffer for 1 hour at room temperature. Secondary antibodies included donkey anti-mouse 488 (1:500) (Thermo Fisher Scientific Cat #: A-21202, RRID: AB_141607), donkey anti-goat 568 (1:500) (Thermo Fisher Scientific Cat #: A-11057, RRID: AB_2534104), and donkey anti-rabbit 647 (1:500). Slides were then rinsed and Hoechst solution (1:10,000) (Invitrogen H3570) was applied for 5 minutes to label nuclei. Finally, slides were rinsed, coverslipped with fluoromount G, sealed with nail polish, and stored at 4° C.
Cultures and columns were fixed with 4% formaldehyde for 35 minutes at room temperature, rinsed with PBS, permeabilized with 0.3% Triton X-100 at room temperature for 20 minutes, blocked with 4% normal horse serum at room temperature for one hour, and again rinsed with PBS. Cultures were then incubated in primary antibody solutions at 4° C. for 16 hours. All cultures and columns were incubated in Hoechst solution (1:1000) (Invitrogen Cat #: H3570) during primary incubation. Subsequently, cultures were rinsed and incubated in appropriate Alexa secondary antibodies (1:500) in the dark at 37° C. for 2 hours. Rat cortical astrocyte planar cultures, extracted rat cortical astrocyte TE-RMSs, human gingiva stem cell-derived planar cultures, and extracted human gingiva stem cell-derived TE-RMSs were incubated in mouse anti-Ezrin (1:100) (Sigma-Aldrich Cat #E8897, RRID: AB_476955), goat anti-GFAP (1:1000) (Abcam Cat #ab53554, RRID: AB_880202), and rabbit anti-Robo2 (1:50) (Novus Cat #NBP1-81399, RRID: AB_11013687) followed by secondary antibodies donkey anti-mouse 488 (Thermo Fisher Scientific Cat #: A-21202, RRID: AB_141607), donkey anti-goat 568 (Thermo Fisher Scientific Cat #: A-11057, RRID: AB_2534104), and donkey anti-rabbit 647 (Thermo Fisher Scientific Cat #: A-31573, RRID: AB_2536183). To verify astrocytic phenotype, human gingiva stem cell-derived astrocyte-like planar cultures were incubated in mouse anti-CD31 (1:100) (Bio-Rad Cat #: MCA1746GA, RRID: AB_2832958) to label endothelial cells, and guinea pig anti-S100B (1:200) (Synaptic systems Cat #: 287 004, RRID: AB_2620025), chicken anti-GFAP (1:1000) (Abcam Cat #: ab4674, RRID: AB_304558), rabbit anti-GLAST (EAAT1) (Abcam Cat #: ab41751, RRID: AB_955879), and mouse anti-glutamine synthetase (Abcam Cat #: ab64613, RRID: AB_1140869) to label astrocytes; secondary antibodies were donkey anti-mouse 488 (Thermo Fisher Scientific Cat #: A-21202, RRID: AB_141607), donkey anti-guinea pig 568 (Sigma Cat #: SAB4600469, RRID: AB_2832959), donkey anti-rabbit 568 (Thermo Fisher Scientific Cat #: A10042, RRID: AB_2534017), and donkey anti-chicken 647 (Jackson Immunoresearch Cat #: 703-605-155, RRID: AB_2340379). Fixed human GMSC-derived TE-RMSs loaded with cortical neuronal aggregates were incubated in mouse anti-human nuclei (1:200) (Millipore Cat #: MAB1281, RRID: AB_94090) to label human astrocytes, rabbit anti-beta III tubulin (TuJ1) (1:500) (Abcam Cat #: ab18207, RRID: AB_444319) to label immature migrating neurons, and goat anti-GFAP (Abcam Cat #ab53554, RRID: AB_880202)) to label astrocytes in TE-RMSs; followed by secondary antibodies donkey anti-mouse 488 (Thermo Fisher Scientific Cat #: A-21202, RRID: AB_141607), donkey anti-rabbit 568 (Thermo Fisher Scientific Cat #: A10042, RRID: AB_2534017), and donkey anti-goat 647 (Thermo Fisher Scientific Cat #A-21447, RRID: AB_2535864). ECM-only columns were stained with rabbit anti-collagen (1:100) (Abcam Cat #: ab34710, RRID: AB_731684) or rabbit anti-laminin (1:500) (Abcam Cat #: ab11575, RRID: AB_298179) followed by secondary staining with donkey anti-mouse 488 (Thermo Fisher Scientific Cat #: A-21202, RRID: AB_141607) and donkey anti-rabbit 568 (Thermo Fisher Scientific Cat #: A10042, RRID: AB_2534017). All cultures and constructs were rinsed following secondary antibody staining. Cultures and constructs in columns were stored in PBS at 4° C. Coverslips containing extracted TE-RMSs were rinsed once in deionized water and mounted onto glass slides with fluoromount G. The edges of the slides were sealed with nail polish and stored at 4° C.
Six days after TE-RMS and control column implantation subjects were anesthetized with Euthasol and fixed via transcardial perfusion with 0.1% heparinized saline followed by 4% paraformaldehyde. Brains were extracted and submerged in formalin for 24 hours. Brains were sagittally blocked, embedded in paraffin, sliced into 8 μm sections, and mounted on slides. Slides were deparaffinized, rehydrated, and underwent heat-induced epitope retrieval in TRIS-EDTA. Slides were then blocked in horse serum for 30 minutes. Primary antibodies were applied in 1× Optimax buffer overnight at 4° C.: mouse anti-human nuclei (1:500) (Millipore Cat #: MAB1281, RRID: AB_94090) to label human astrocytes, goat anti-doublecortin (DCX) (1:500) (Novus Cat #: NBP1-72042, RRID: AB_11019667) to label immature migrating neurons, and either chicken anti-GFAP (1:1000) (Abcam Cat #: ab4674, RRID: AB_304558) or rabbit anti-collagen (1:100) (Abcam Cat #: ab34710, RRID: AB_731684). Slides were then rinsed in PBS/Tween and incubated in Alexa secondary antibodies (1:500) in 1× Optimax buffer for 1 hour at room temperature: donkey anti-mouse 488 (Thermo Fisher Scientific Cat #: A-21202, RRID: AB_141607), donkey anti-goat 568 (Thermo Fisher Scientific Cat #: A-11057, RRID: AB_2534104), donkey anti-chicken 647 (Jackson Immunoresearch Cat #: 703-605-155, RRID: AB_2340379), or donkey anti-rabbit 647 (Thermo Fisher Scientific Cat #: A-31573, RRID: AB_2536183). Slides were rinsed and Hoechst solution (1:10,000) (Invitrogen H3570) was applied for 5 minutes. Slides were rinsed, coverslipped with fluoromount G, sealed with nail polish, and stored at 4° C.
Planar-cultured GMSCs or astrocytes induced from GMSCs were harvested and whole cell lysates were prepared by incubation with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz) supplemented with a cocktail of protease inhibitors (Santa Cruz) and the total protein concentrations were determined using bicinchoninic acid (BCA) method (BioVision). Then 30 μg of proteins per well were subjected to SDS-polyacrylamide gel electrophoresis before being electroblotted onto a 0.2 μm nitrocellulose membrane (GE Healthcare). After blocking with 5% nonfat dry milk in TBST (25 mmol/L Tris, pH, 7.4, 137 mmol/L NaCl, 0.5% Tween20), membranes were incubated overnight at 4° C. with following primary antibodies: GFAP (1:1000, ab53554, Abcam), glutamine synthetase (1:1000, ab64613, Abcam), or GAPDH (1:2000, #5174, Cell Signaling) as loading control. After extensively washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz) and blot signals were developed with ECL™ Western Blotting Detect Reagents (GE Health Care) and scanned using Amersham Imager 680.
Cultures and constructs were routinely imaged for observation using phase contrast and epifluorescence microscopy on a Nikon Inverted Eclipse Ti-S microscope with digital image acquisition using a QiClick camera interfaced with Nikon Elements Basic Research software (4.10.01). Epifluorescence images for analysis were captured using a Nikon Eclipse Ti-S inverted epi-fluorescent scope outfitted with an Andor Zyla sCMOS 5.5 megapixel camera interfaced with Nikon Elements Basic Research software (4.10.01) with either a 10× (Plan Apo Lambda 10×, n.a. 0.45) or 20× (Plan Apo Lambda 10×, n.a. 0.75) objective. All images acquired for comparative analyses were captured with identical acquisition settings. Samples were also fluorescently imaged using a Nikon A1Rsi Laser Scanning Confocal microscope with a 10×, 20×, or 60× objective (CFI Plan Apo Lambda 10×, n.a. 0.45; 20×, n.a. 0.75; or 60× Oil, n.a. 1.40).
Image processing and analyses were performed using the freely available FIJI (Fiji Is Just ImageJ) software platform85. Values reported in the Results section are mean±SEM, unless otherwise noted. Statistical testing was performed in GraphPad Prism 8 for Windows 64 bit. Due to the obvious differences between protoplasmic and TE-RMS astrocytes, blinding was not possible. Therefore, we minimized potential bias by maximizing automation via design and application of macros for automated image processing and analyses. All Nikon nd2 files were imported into FIJI via the Bioformats function and each channel was split into an individual grayscale Tiff. Background subtraction was applied to all images using the rolling ball method with a diameter of 100 pixels.
To compare endogenous rat RMS glial tube and protoplasmic astrocytes as summarized in
To compare TE-RMS astrocytes and astrocytes from planar sister cultures as summarized in
To compare migrating neurons in our in vitro migration assay, the Hoechst channel for each image was first converted to a binary mask in FIJI. For the TE-RMS group, the human nuclei channel was also converted to a binary mask and then subtracted from the corresponding Hoechst channel using the Image Calculator “Subtract” function in FIJI to remove TE-RMS nuclei and isolate signal from migrating rat neurons. Partially removed human nuclei still present after the subtraction function (evident as open circles) were removed via the Binary>Open function in FIJI. Particle counts were unreliable due to inconsistencies resolving adjacent and overlapping nuclei, therefore the total nuclear area was measured via the “Analyze Particles” function in FIJI. For each assay, measurements were taken from two ROIs spanning the width of the inner lumen of the microcolumns (300 μm). The first ROI extended 1 mm from the edge of the neuronal aggregate, and the second ROI extended an additional 2.5 mm from the end of the first (extending from 1 to 3.5 mm from the edge of the aggregate). Mean nuclear areas were compared via one-way ANOVA with Bonferroni adjustment for multiple comparisons. A Log transform for all values was performed to meet assumptions of normality and equal variance. Neurite extension from neuronal aggregates in collagen and collagen/laminin columns was measured from the edge of the aggregate to the end of the longest Tuj1-positive neurite using the line segment measuring tool in FIJI. The analyst was blinded to ECM composition. Statistical comparisons were not performed after removing the blind because collagen-only columns did not exhibit any measurable neurite outgrowth.
Primary cortical astrocytes were harvested from Sprague-Dawley rat pups at postnatal day 0-1. Following dissociation (described previously [27]), astrocytes were cultured in DMEM/F12 medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin antibiotics in a cell culture incubator maintained at 37° C. and 5% CO2. Astrocytes were passaged at 80% confluency. Astrocytes between passages 4-10 were utilized for fabrication of TE-RMSs and planar astrocyte cultures.
TE-RMSs were fabricated in hydrogel micro-columns made of 3% agarose. Fabrication of micro-columns has been described previously [27,30,31]. Briefly, warm agarose dissolved in Dulbecoo's phosphate-buffered saline (DPBS) was drawn into a capillary tube (inner diameter=701 microns) with a needle (diameter=300 microns) in the center. Following agarose cooling and capillary tube removal, the micro-column was pushed off the needle into PBS, effectively creating micro-columns with an inner diameter of 300 microns and an outer diameter of 701 microns. Previous experiments have determined these dimensions to be optimal for astrocyte bundling and TE-RMS formation [26].
Fabrication of TE-RMSs has been recently described [29]. Micro-columns were cut to a 4 mm length and the inner lumen was seeded with 1 mg/mL rat tail type 1 collagen (Corning Catalog #354236) diluted in in serum free co-culture media consisting of Neurobasal medium supplemented with 2% B27, 1% G-5, 0.25% L-glutamine, and 1% penicillin-streptomycinastrocyte culture media. Columns were placed in an incubator at 37° C. and 5% CO2 for around 3 h until collagen completely polymerized to coat the inner walls of the micro-columns. During the collagen polymerization period, a flask of 80% confluent astrocytes was passaged and cells were resuspended in serum free co-culture media consisting of Neurobasal medium supplemented with 2% B27, 1% G-5, 0.25% L-glutamine, and 1% penicillin-streptomycin. Following complete collagen polymerization, micro-columns were seeded with astrocytes in co-culture media at a density of around 1 mil-lion cells/mL. Columns were then returned to the incubator at 37° C. and 5% CO2 and one hour later the outer edges of the columns were reinforced with 1 mg/mL collagen. Columns were incubated for another hour at 37° C. and 5% CO2, dishes were flooded with warm co-culture media, and then columns were returned to the incubator at 37° C. and 5% CO2. Under these conditions, astrocytes bundle together with collagen and self-assemble into cords of longitudinally aligned astrocytes with bidirectional processes to form TE-RMSs. All experiments herein investigated TE-RMSs at 24 h following astrocyte seeding into microcolumns. For planar astrocyte samples, a flask of 80% confluent astrocytes was passaged, cells were resuspended in co-culture media, and were plated on top of polymerized 1 mg/mL collagen in a 12-well plate at a density of around 1 million cells/mL in co-culture media. For 100× fluorescent imaging of planar astrocytes, cells were cultured in 12-well plates on top of poly-L-lysine (PLL) and collagen-coated glass coverslips. For scanning electron microscopy (SEM) imaging, planar astrocytes were cultured in 24-well plates on top of PLL-coated glass coverslips.
Following TE-RMS formation, constructs were extracted from micro-columns onto poly-1-lysine glass slides for immunocytochemical analyses as previously described [29]. Planar astrocyte cultures and extracted TE-RMSs were fixed with 4% paraformaldehyde for 30 min at room temperature. Following rinsing with PBS, cultures and constructs were permeabilized with 0.3% Triton X-100 and blocked with 4% normal horse serum for one hour at room temperature and again rinsed with PBS. Cultures and constructs were incubated in the primary antibody goat anti-glial fibrillary acidic protein (GFAP) (1:1000) (Abcam Cat #ab53554, RRID: AB_880202) overnight at 4° C. Cultures were then rinsed and incubated in the Alexa secondary antibody donkey anti-goat 568 (1:500) (Thermo Fisher Scientific Catalog #: A-11057, RRID: AB_2534104) and Hoechst solution (1:1000) (Invitrogen H3570) in the dark for two hours at 37° C.
Five adult Sprague-Dawley rat brains from an archival tissue bank were selected for analysis of the endogenous RMS. Brains were formalin-fixed paraffin-embedded (FFPE) and sagittally sliced into 8-micron thick sections and mounted onto slides. Slides were deparaffinized, rehydrated, and placed in Tris-EDTA for heat-induced epitope retrieval. Slides were blocked in normal horse serum for 30 min and the primary antibody goat anti-GFAP (1:1000) (Abcam Cat #ab53554, RRID: AB_880202) was diluted in 1× Optimax buffer and applied overnight at 4° C. Slides were washed in PBS Tween and incubated in the dark with the Alexa secondary antibody donkey anti-goat 568 (1:500) (Thermo Fisher Scientific Catalog #: A-11057, RRID: AB_2534104). Slides were then rinsed and incubated in Hoechst solution (1:10,000) (Invitrogen H3570) for 5 min and then rinsed again. Cover slips were mounted with fluoromount G and stored at 4° C.
Fluorescent images of cultures, constructs, and brain slices were obtained using a Nikon A1Rsi Laser Scanning Confocal microscope with a ×10, ×20, ×60, or ×100 objective (CFI Plan Apo Lambda×10, n.a. 0.45; ×20, n.a. 0.75; ×60 Oil, n.a. 1.40; ×100, n.a. 1.45). Scanning electron microscope experiments were carried out at the CDB Microscopy Core (Perelman School of Medicine, University of Pennsylvania). Samples were washed three times with 50 mM Na-cacodylate buffer, fixed for 2 h with 2% glutaraldehyde in 50 mM Na-cacodylate buffer (pH 7.3), and dehydrated in a graded series of ethanol concentrations through 100% over a period of 2.5 h. Dehydration in 100% ethanol was done three times. Dehydrated samples were incubated for 20 min in 50% HMDS in ethanol followed by three changes of 100% HMDS (Sigma-Aldrich Co.) and followed by overnight air-drying as described previously [32]. Then samples were mounted on studs and sputter coated with gold palladium. Specimens were observed and photographed using a Quanta 250 FEG scanning electron microscope (FEI, Hillsboro, OR, USA) at 10 kV accelerating voltage.
Image analyses were performed using FIJI (Fiji Is Just ImageJ) software [33]. Statistical testing was performed in GraphPad Prism 9 for macOS. 20× fluorescent confocal images were utilized for quantification of all nuclear and cytoskeletal measurements. Fluorescent images were imported into FIJI as Nikon nd2 files, split into two channels (Hoechst and GFAP), z-stacked and compressed, and underwent background subtraction (rolling ball method; 50 pixel diameter). In in vitro planar astrocyte and TE-RMS images, all cells with non-overlapping nuclei were quantified. Across TE-RMS samples, we estimate that about 50% of TE-RMS astrocytes were excluded from analysis due to the appearance of overlap-ping nuclei. In in vivo images, protoplasmic and RMS astrocytes were quantified only if they possessed visible, non-overlapping nuclei and clear GFAP process extensions. Endogenous RMS astrocytes were identified within the dense, distinct meshwork of GFAP processes extending from the lateral ventricle to the olfactory bulb, as reliably identified previously by our laboratory and others [29,34]. Protoplasmic astrocytes were identified as astrocytes outside of this distinct structure. To measure the long and short nuclear axes (
We used fluorescence immunocytochemistry (ICC) to label nuclei (Hoechst) and the intermediate filament protein glial fibrillary acidic protein (GFAP) in planar astrocyte (n=6) and TE-RMS (n=9) samples to analyze the overall complexity of the cytoskeletal arrangement of in vitro cultures. For each sample, a single 20× fluorescent confocal image was used for quantification. We quantified the number of main processes, number of branch points, and angle of each main process across planar astrocytes (n=225 cells) and TE-RMS astrocytes (n=275 cells). Example representative images of planar (
Endogenous Protoplasmic Astrocytes have a More Complex and Varied Cytoskeletal Arrangement Compared to Endogenous RMS Astrocytes in Rat Brain
To compare our in vitro cytoskeletal findings to the rat brain in vivo, we applied fluorescence immunohistochemistry (IHC) techniques to label GFAP and Hoechst in sagittally sectioned (
We performed nuclear measurements in the same planar (
To compare our in vitro nuclear measurements to astrocytes in the endogenous rat brain, we performed nuclear measurements in the same endogenous protoplasmic and RMS astrocytes (
High magnification (100×) fluorescent confocal imaging was conducted to examine the differences between nuclear shape and intermediate filament arrangement of planar (
To further examine the fine structural differences in nuclear morphology and cytoskeletal arrangement, planar astrocytes (
We also directly compared cytoskeletal and nuclear measurements of TE-RMS astrocytes and endogenous RMS astrocytes (data depicted in Table 1). Four two-tailed, nested t-tests were performed on measurements of main processes, number of branch points, angle of main processes, and nuclear aspect ratios. To further compare these parameters, we also present descriptive statistics to report the mean and standard deviation (SD). Here, mean±SD values for TE-RMS and RMS astrocytes were calculated by grouping all cells from each condition (Table 1). Degrees of freedom (DF), t values, and p values are from the nested t-test analyses run between TE-RMS and RMS groups. This testing showed that the number of main processes (t=0.6326, df=12, p=0.5388) and the number of branch points (t=0.1509, df=12, p=0.8825) were statistically equivalent between the TE-RMS and endogenous RMS astrocytes. However, there were modest differences in the angle of the main processes (t=2.861, df=12, p=0.0143) and the nuclear aspect ratio (t=2.490, df=12, p=0.0284) between astrocytes in the TE-RMS and astrocytes in the endogenous RMS. Interestingly, these analyses revealed that TE-RMS astrocytes were slightly more aligned and presented more oblong nuclei than endogenous RMS astrocytes.
The TE-RMS is the first engineered biomimetic astrocytic microtissue that is designed to promote sustained neuroblast redirection into distal neuron-deficient brain regions. The TE-RMS may also be a useful platform to investigate the mechanisms of neuronal migration, cell-cell communication, and differentiation in vitro, which may also feature human cells to provide insights into human neurogenesis and migration. The self-assembly of the TE-RMS in biomaterial microcolumns leads to profound morphological changes in astrocytes. We demonstrate that when astrocytes are put into specific 3D conditions with all other conditions kept consistent, they undergo rapid process/cytoskeletal remodeling and nuclear elongation. We have previously demonstrated that a specific angle of curvature of hydrogel microcolumns is required for proper TE-RMS formation [26]. These unique bio-material conditions initiate a fundamentally different program in these cells that results in dramatic structural changes. These morphological features, which have not previously been described in astrocytes in vitro, do not require growth factors or media changes-rather, they are caused solely by the geometric and topographical feature of the 3D environment that the cells are exposed to. The resulting novel astrocytic phenotype warrants further investigation, for instance as a tool to investigate astrocyte cytoskeletal-nuclear dynamics and cell-environmental interactions that would otherwise not be possible and thereby expanding the capabilities of the TE-RMS as a tool for scientific discovery. Further, we demonstrate that the unique cytoskeletal arrangement and nuclear shape of TE-RMS astrocytes mimics that of astrocytes in the endogenous rat RMS. Whereas planar astrocyte cultures have similar morphology to endogenous protoplasmic astrocytes, the bidirectional processes and elongated nuclei that are characteristic of TE-RMS astrocytes mimic that of RMS astrocytes. These findings augment previous work demonstrating that the TE-RMS mimics the general structure and protein expression of the endogenous RMS, highlighting this technology as the first biomimetic astrocytic microtissue that replicates the structural and functional features of the endogenous pathway for neuroblast delivery in the mature brain.
While the number of main processes and branch points were equivalent between TE-RMS and endogenous RMS astrocytes, we reported small, yet statistically significant, differences in cytoskeletal alignment and nuclear shape measurements between TE-RMS and endogenous RMS astrocytes (as summarized in Table 1). The TE-RMS nuclear aspect ratio of 2.56±0.98 (mean±SD) was higher compared to that of the endogenous RMS nu-clear aspect ratio of 2.23±0.67 (mean±SD). Additionally, the main processes of TE-RMS astrocytes were more aligned with the longitudinal axes of the cells with a deviation of 12.42±16.42 (mean±SD) degrees compared to endogenous RMS astrocytes which had a deviation of 18.63±19.39 (mean±SD) degrees from the longitudinal axes. As these differences highlight, the phenomena of elongated nuclei and aligned cytoskeleton appear to be greater in the TE-RMS compared to that of in vivo RMS astrocytes. There are several possible explanations for these differences. First, our in vitro TE-RMS system is only around 24 h old at the time of these analyses. We predict that this system will continue to mature as it is cultured in vitro to further emulate in vivo RMS morphology. Additionally, our TE-RMS system lacks in vivo cues that could subtly affect astrocyte morphology, including the presence of vasculature and other cell types, the presence of various types of ECM and soluble factors, and signals from migrating neuroblasts.
Astrocytes are mechanosensitive, meaning that they can sense and respond to mechanical cues in their surrounding environment [35]. Recent research has begun to elucidate the mechanisms of astrocyte mechanosensation and the significance of this property for physiological astrocyte function in vivo [36,37]. Additionally, nuclear pore complexes, which fuse the two layers of the nuclear envelope, are mechanosensitive thereby making the nucleus itself sensitive to changes in cellular mechanical forces [38-40]. The four main contributing factors to the shape of a cell's nucleus are cytoskeletal forces, the thickness of the nuclear lamina, level of chromatin compaction, and activity of proteins that control chromatin conformation [41]. Here, we demonstrate significant nuclear elongation of astrocytes following TE-RMS formation, with some astrocytes having a nuclear aspect ratio as high as 8. This extent of TE-RMS nuclear elongation, which has never been reported in planar astrocyte cultures but mimics astrocyte morphology in the endogenous RMS, hap-pens remarkably quickly following astrocyte introduction to 3D conditions. The unique cytoskeletal changes that are concomitant with nuclear elongation during TE-RMS formation indicates that the cytoskeleton is involved in executing these nuclear morphological changes. Cytoskeletal forces propagate to the nucleus through the linker of nucleoskeleton and cytoskeleton (LINC) complex which connects the cytoskeleton to the nuclear envelope [41]. The LINC complex directly contacts cytoskeletal actin filaments and indirectly contacts intermediate filaments through cyto-linker proteins and microtubules through motor proteins to allow for mechanotransduction of extracellular forces to the nucleus [41]. Cytoskeletal forces regulate nuclear size, shape, orientation, and movement within a cell [41]. For example, Versaevel and colleagues demonstrated that the shape and orientation of endothelial cell nuclei can be controlled by compressive forces exerted by actin filaments [42]. Topographical curvature can also control the shape of epithelial cells in vitro, including the orientation and distribution of epithelial cell nuclei and F-actin [43]. Additionally, intra-ocular pressure has been shown to modulate the astrocyte cytoskeleton and nuclear shape in the optic nerve of mice [44]. In the TE-RMS, astrocyte processes align in parallel inside of microcolumns, and they work in conjunction with each other to stretch out the entire cell including the nucleus which can sense these mechanical changes. As cells stretch, the forces acting upon the nuclei become different than the forces acting upon cells in 2D conditions that have radial processes. While we hypothesize that cytoskeletal forces are the predominant mechanism leading to greater nuclear eccentricity, there could be additional factors that are causing these nuclear shape changes. For example, Kalinin and colleagues recently demonstrated that modulation of chromatin compaction with valproic acid treatment leads to changes in astrocyte nuclear shape [45]. We are currently investigating how these biomaterial conditions that induce TE-RMS formation affect the structure of the astrocyte nuclear lamina and chromatin arrangement to elucidate how they may contribute to astrocyte nuclear elongation.
Advanced imaging technologies provide an in-depth view of the astrocyte structural changes that result from TE-RMS formation. High magnification fluorescent imaging (
Herein, we reveal critical details describing the unique astrocyte structure that emerges from exposure to specific 3D biomaterial conditions. It is well known that mechanical changes in nuclear morphology are often accompanied by changes in chromatin organization and gene expression which then lead to downstream alterations in cell activity and signaling [41,51-54]. However, there is limited research on how mechanically altering nuclear shape affects gene expression regulation and cell behavior in astrocytes specifically [44,45]. Overall, we performed a thorough structural evaluation of the intermediate filament cytoskeleton and nuclear morphology in TE-RMS astrocytes, revealing a novel astrocyte phenotype that has not previously been described in vitro. The TE-RMS offers a powerful platform to investigate critical relationships between biomaterial surface cues and astrocyte behavior and function.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Further, it should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow.
The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/197,007 filed Jun. 4, 2021, the content of which is incorporated by reference herein in its entirety.
This invention was made with government support under NS103253 awarded by the National Institutes of Health, 1845298 awarded by the National Science Foundation, W81XWH-16-1-0796 awarded by the U.S. Army, Merit Award 101-BX003748 and IK2-RX003376 provided by the United States Government as represented by the Department of Veterans Affairs. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/32146 | 6/3/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63197007 | Jun 2021 | US |
