Patent Application
20240009248
The present disclosure relates to methods of generating neural progenitor cells with a spinal cord identity from starter neural progenitor cells that express Sox2, Pax6, Nestin and at least one of the brain markers (such as Otx2, Foxg1 or Gbx2), or from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), as well as to compositions comprising said cells or components for making said cells and uses thereof.
Spinal cord injuries (SCIs), frequently the result of falls or road traffic accidents, have devastating long-term physical, social, and financial impacts on patients and their families (Furlan et al., 2011; Sekhon and Fehlings, 2001). Mechanical compression and stretching of the spinal cord at the time of injury results in axonal damage as well as the loss of neurons and glia due to necrosis and apoptosis (Baptiste and Fehlings, 2006; Rowland et al., 2008; Tator et al., 1993). The loss of these critical cells prevents the transmission of neural signals from the brain to the rest of the body. Without these signals, an individual's ability to perform everyday activities such as walking, grasping and controlling bowel/bladder function is compromised. Cell transplantation with neural stem/progenitor cells to repair and regenerate the spinal cord is a promising treatment strategy for SCI (Khazaei et al., 2017; Nagoshi et al., 2018). A wide range of neural progenitor cells at different developmental stages have been studied as potential treatments. These studies have revealed multiple mechanisms underlying recovery, including cell replacement and trophic support. These mechanisms are believed to be partly determined by the source and developmental stage of the transplanted cells.
The generation of stem cells expressing distinct neural phenotypes is a complex multi-stage process, which starts during early neural tube development with invagination of the neuroectodermal layer to form neuroepithelial stem/progenitor cells (NPCs). NPCs in the rostral neural tube with an anterior identity ultimately form forebrain forebrain NPCs (fbNPC). Conversely, NPCs exposed to a temporally and spatially different gradient of morphogens gradually mature and caudalize, leading to the formation of the mid- and hind-brain. After further temporospatial maturation, these cells become further caudalized and then ventralized to form spinal cord NPCs (spNPCs)(Gifford et al., 2013). fbNPC and spNPCs are temporally and spatially located at different spectrums of this continuum (
Numerous central nervous system (CNS) disorders involve loss of key neuroglial cell populations. Cell-based regenerative approaches have therefore emerged as promising strategies to improve long-term outcomes for patients. Although early research employed primary donor cells, limited supply and the relative inaccessibility of CNS tissue made this approach infeasible for widescale therapy. As a result, the field has gravitated towards clinically relevant pluripotent stem cells (PSCs) as these cells can differentiate into any somatic human cell type.
Embryonic stem cells (ESCs) are PSCs capable of developing into the three primary germ cell layers in embryos. Studies of ESCs formed the basis for a substantial body of knowledge around stem cell culture and cell differentiation pathways. Unfortunately, limited availability and potential ethical concerns remain a challenge. The discovery of induced pluripotent stem cells (iPSCs) and associated culture protocols led to a resurgence of regenerative therapy research. iPSCs are reprogrammed cells derived from any somatic cell of any patient and are capable of forming all three germ cell layers as well as early-stage organoids. Their postnatal derivation eliminates ethical concerns and the potential to produce iPSCs as autologous therapies significantly reduces the risk of immune rejection.
While numerous cell types, including mesenchymal stem cells, olfactory ensheathing cells and Schwann cells have been studied for central nervous system (CNS) regeneration1, neural progenitor cells (NPCs), also known as neural stem cells, are the most promising as they are tripotent cells capable of differentiating into neurons, oligodendrocytes, or astrocytes; the primary cell types of the brain and spinal cord. To date, NPCs have been investigated for the treatment of numerous CNS conditions, including spinal cord injury (SCI), Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and stroke2-8.
The regional identity of brain and spinal cord NPCs is established early in neurodevelopment (
The most anterior/rostral fate is the first regional identity that is established9. These cells express transcription factors such as Otx2, FoxG1, DIx2, Tbr1, and Tbr210,11. Foxg1, is expressed continuously in the postnatal and adult hippocampal dentate gyrus (DG)12. Following rostral neural fate acquisition, the caudal identities will be determined by positional cues supplied by spatial gradients of patterning factors such as Wnt, FGFs, DKK1, and FRZB. These cells in the spinal cord express transcription factors, including HoxA2, HoxA3, and HoxB313 (
As in vivo, neural progenitors derived from PSCs in vitro first acquire rostral identity by default14, and this primitive identity can be converted to more caudal fates by various cues such as retinoic acid (RA), WNTs or FGFs , thereby mimicking the in vivo situation.
Unfortunately, a significant challenge remaining in NPC research is the optimization of graft-host integration to maximize functional recovery. Regional identity has emerged as a key consideration in the use of NPCs as therapy. Several studies have demonstrated that matching cell identity to the target tissue significantly improves graft-host integration and cell survival15,16. For diseases predominantly in the spinal cord, such as SCI and ALS, this suggests a need to establish spinal cord-specific NPC lines. Classic protocols to derive pure NPC populations from PSCs generate brain-identity cells. Indeed, studies utilizing these brain identity NPCs found that they differentiate poorly into important populations such as spinal V2a interneurons and spinal motor neurons. Additionally, their integration is impaired by the introduction of foreign cortical populations, including excitatory pyramidal neurons, corticothalamic glutamatergic neurons, and cholinergic neurons. Regeneration of the corticospinal tract (CST) has also been observed following transplantation with adult spinal NPCs (spNPCs) but not forebrain NPCs (fbNPCs)17.
The accumulating evidence in favour of regional-identity matching highlights a notable gap in current regenerative spinal research. Namely, most existing protocols to generate NPCs from human PSCs (hPSCs) produce cells expressing a forebrain and/or midbrain identity including transcription factors like Otx2, FogG1, Six3, and DIx218-21. Developing a method of generating NPCs with a spinal identity is desirable and may improve cell integration and survival in the spinal cord niche.
Generation of human induced pluripotent stem cells (hiPSCs) from human peripheral blood or any somatic cells, provides a convenient and minimally-invasive method of obtaining patient-specific iPSCs. Described herein is a reproducible protocol for generation of spinal cord identity neural progenitor cells (spNPCs) from starter neural progenitor cells that express Sox2, Pax6, Nestin and at least one of the brain markers (such as Otx2, Foxg1 or Gbx2), or human pluripotent stem cells (hPSCs). The inventors have for example established a culture system and use a serum-free medium and a RAR agonist such as retinoic acid (RA), to differentiate iPSCs into neural progenitor cells (NPCs) with spinal cord identity.
spNPCs made using a method described herein can be characterized by immunocytochemistry staining and/or RT-qPCR analysis.
spNPCs can terminally differentiate into spinal cord specific neuronal cell types like ventral motor neurons and spinal interneurons, Renshaw cells, paragriseal, interstitial and propriospinal interneurons. These neuronal cell types cannot be generated by brain identity (e.g. unpatterned) NPCs. On the other hand brain NPCs can terminally differentiate to neuronal cell types of the brain like cortical, subcortical, or deep nuclear neurons, excitatory pyramidal neurons, Calbindin or CART expressing neurons, corticothalamic glutamatergic neurons and cortical cholinergic neurons that cannot be generated by spinal identity NPCs.
Starter neural progenitor cells (NPCs) that express Sox2, Pax6, Nestin and at least one of the brain markers (such as Otx2, Foxg1 or Gbx2), or PSCs such as hPSCs, hiPSCs and ESCs can be used after they have been differentiated to NPCs. hPSCs, which were used in Examples 1 and 2, expressed pluripotency-associated genes Oct4, Sox2, and Nanog before NPC differentiation. After differentiation, spNPCs expressed general NPC markers (Nestin, Sox2, and reduced Pax6) and also regional identity markers (e.g. Hox genes). It is demonstrated herein that spNPCs generated can be expanded in a neurosphere suspension system or monolayer cultures. Like other NPCs with other regional identities, spNPCs were able to differentiate into cells expressing cellular markers for neurons (e.g. Fox3 and/or β-III-tubulin), astrocytes (e.g. GFAP and/or S100b), and oligodendrocytes (e.g. O1 and/or O4 and/or Olig2 and/or Olig1). Starter NPCs such as unpatterned NPCs have a higher level of brain markers and lower level of spinal markers (e.g. Hox genes) than spinal identity NPCs. For example, spinal NPCs do not express detectable level of (by Immune-staining) of brain markers.
As shown further herein, whole-cell patch clamp recording revealed that neurons differentiated from fbNPCs and spNPCs, exhibited electrophysiological properties of neurons, including action potentials.
A first aspect of the invention comprises a method of producing spinal identity neural progenitor cells (spNPCs), the method comprising:
When starting with unpatterned NPCs, the unpatterned NPCs can be primed.
A second aspect of the invention comprises a method of priming unpatterned NPCs to stay in an ectodermal cell fate, the method comprising:
Another aspect of the invention comprises a method of producing spNPCs from unpatterned NPCs, the method comprising:
Another aspect of the invention comprises a method of producing spNPCs from induced pluripotent stem cells (iPSCs), the method comprising:
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
An embodiment of the present disclosure will now be described in relation to the drawings in which:
Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. For example, the term “a cell” includes a single cell as well as a plurality or population of cells. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art (see, e.g. Green and Sambrook, 2012).
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus for example, a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
As used in this application and claim(s), the word “consisting” and its derivatives, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
The terms “about”, “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.
As used herein, the term “pharmaceutically acceptable carrier” or variations thereof is intended to include any and all solvents, media, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration and for use with cells. Optional examples of such carriers or diluents include, but are not limited to, buffered saline, culture media, Hanks' Balanced Salt solution, ringer's solutions, and 5% human serum albumin and bovine serum albumin (BSA).
The term “neural progenitor cell” or variations thereof also referred interchangeably as neural stem cell (NSC), neural precursor cells (NPC), neural stem progenitor cells (NSPCs), neuroepithelial stem/progenitor cells (NPC) or, neuroectodermal cells (NECs), as used herein includes neural cells that express Sox2, Pax6 and Nestin and are tripotent and differentiable to neurons, astrocytes or oligodendrocytes.
The term “neural progenitor cell with a spinal cord identity” or “spNPC” refers to neural progenitor cells that can terminally differentiate to spinal cord specific neuronal cell types like ventral motor neurons and spinal interneurons, Renshaw cells, paragriseal, interstitial and propriospinal interneuron cells, and which express elevated levels of spinal cord genes such Hox genes such as Hox A, B, C or D, 1-10 (e.g. A4, A5, B4, C4) in a higher amount than brain NPCs and express lower amounts of brain markers for example Gbx2, Otx2, FoxG1, Emx2 and/or Irx2 as well as Pax6 as compared to brain NPCs. Methods for producing spNPCs in vitro are provided herein.
Brain NPCs can differentiate to neuronal cell types of the brain like cortical, subcortical, or deep nuclear neurons, excitatory pyramidal neurons, Calbindin or CART expressing neurons, corticothalamic glutamatergic neurons and cortical cholinergic neurons that cannot be generated by spinal neurons.
The term “unpatterned NPCs” or variations thereof as used herein refers to NPCs that have a rostral identity, and are not caudalized and/or ventralized. Unpatterned NPCs are primitive or definitive NPCs which are not yet being treated with any patterning factors like RA or Shh (and its agonists). Unpatterned NPCs express Pax6, Nestin and Sox2 and at least one of the brain markers OTX2, FOXG1, and GBX2. The level of expression of Gbx2, Emx2 and Irx2 is lower in un-patterned NPCs as compared to mid-brain identity NPCs, and the level of expression of Hox genes (like A4, A5, B4, C4) are lower in un-patterned NPCs as compared to spinal cord identity NPCs. Unpatterned NPCs can be referred to as NPCs with forebrain identity (fbNPCs), NPCs with front brain identity, or anterior brain NPCs. All of these terms refer unpatterned NPCs. They can be used interchangeable throughout this disclosure.
The term “posteriorized NPCs” or variations thereof as used herein refers to tripotent neural progenitor cells with the same differentiation profile as unpatterned NPCs. The ability to form neurospheres and the proliferation rate of posteriorized NPCs are marginally higher than unpatterned NPCs. Treatment of unpatterned NPCs with a FGF8 agonist, optionally FGF8 under specific concentrations and time periods as described herein, results in posteriorization of the cells. Posteriorized NPCs express more Hox genes, such as HoxA4 and/or HoxA5, and have reduced expression of at least one of the brain markers such as DIx2, Six3, LmxA1, Gbx2, Otx2 and/or FoxG1 compared to unpatterned cells.
The term “EGF-L7 agonist” or variations thereof as used herein means EGF-L7, preferably human EGF-L7, (Accession number Ensembl:ENSG00000172889, MIM:608582) as well as active fragments, fusions and active splice variants thereof as well as any compound or combination of compounds, natural or synthetic, that simultaneously or in combination inhibits Notch signaling, activates EGFR (EGF receptor), inhibits ICAM-1 expression and enhances the inhibition of NF-κB activation. Combinations of molecules that activate a corresponding set of pathways can also be used instead of EGF-L7. For example a combination DLK1 or DAPT (to inhibit Notch), NSC228155 or betacelluin (to activate EGFR), A-205804 (to inhibit ICAM-1), and Bortezomib and Bardoxolone Methyl (to inhibit NF-KB) can be used. The term “EGF-L7” as used herein means EGF-L7, preferably human EGF-L7, (Accession number Ensembl:ENSG00000172889, MIM:608582) as well as active fragments, fusions and active splice variants thereof that simultaneously inhibits Notch signaling, activates EGFR (EGF receptor), inhibits ICAM-1 expression and enhances the inhibition of NF-KB activation.
The term “EGF receptor agonist” as used herein means any compound or combination of compounds, natural or synthetic that binds and/or induces EGF receptor (also known as EGFR; ErbB-1; HER1) tyrosine kinase activity, and includes without limitation EGF, and EGF analogs as well as heparin binding EGF (HB-EGF), transforming growth factor (TGF) amphiregulin (AR) and betacellulin. Also included are EGFR activators such as NSC228155.
The term “EGF” or variations thereof as used herein refers to epidermal growth factor. EGF, for example human EGF, includes active fragments, fusions and splice variants (e.g. fragments, fusions and splice variants that activate EGF receptor) can be obtained from various commercial sources such as Cell Sciences®, Canton, Mass., USA, Invitrogen Corporation products, Grand Island N.Y., USA, ProSpec-Tany TechnoGene Ltd. Rehovot, Israel, and Sigma, St Louis, Mo., USA.
The term “FGF2 agonist” or variations thereof as used herein means any compound or combination of compounds, natural or synthetic, that binds the FGF receptors that are bound by FGF2, such as FGFR1, FGFR2, FGFR3 and FGFR4, and includes FGF2, active fragments, fusions and splice variants thereof or molecules with similar function such as SUN11602 or combinations thereof.
The term “fibroblast growth factor 2” or “FGF2” or variations thereof (also known as bFGF, basicFGF, FGFb, or FGF-beta as well as heparin binding growth factor 2) as used herein refers to a member of the fibroblast growth factor family. FGF2, for example human FGF2, includes active fragments, fusions and splice variants(e.g. fragments, fusions and splice variants that activate FGF receptors that are bound by FGF2) can be obtained from various commercial sources such as Cell Sciences.RTM., Canton, Mass., USA, Invitrogen Corporation products, Grand Island N.Y., USA, ProSpec-Tany TechnoGene Ltd. Rehovot, Israel, and Sigma, St Louis, Mo., USA.
The term “FGF8 agonist” or variations thereof as used herein means any compound or combination of compounds, natural or synthetic, that binds the FGF receptors that are bound by FGF8, such as FGFR1, FGFR2, FGFR3 and FGFR4, and includes FGF8, active fragments, fusions and splice variants thereof or molecules with similar function such as FGF9, or FGF17 or active fragments, fusions and splice variants thereof (e.g. fragments, fusions and splice variants that activate FGF receptors that are bound by FGF8) as well as combinations thereof.
The term “FGF8” or variations thereof as used herein means FGF8 A, B or E isoform, referred to example as FGF8a, FGF8b or FGF8e and includes all naturally occurring or synthetic variants thereof, as well as mammalian FGF8 and in particular human FGF8, active fragments, fusions and splice variants thereof as well as combinations thereof. FGF8 is also called androgen-induced growth factor (AIGF).
The term “740Y-P”, “analog of 740Y-P” or variations thereof refer to a cell-permeable phosphopeptide activator of P13K RQIKIWFQNRRMKWKKSDGGYMDMS where Tyr-21 =pTyr) and analogs thereof and includes any compound or combination of compounds, natural or synthetic, that activates P13K kinase including for example erucic acid or active fragments, fusions and splice variants thereof as well as combinations thereof. 740Y-P can also be replaced with high doses of FGF2 or an FGF2 agonist, for example FGF2 at a concentration of greater than 20 ng/ml up to 400 ng/ml for example at least or about 200 ng/mL. 740Y-P is commercially available and can be purchased for example from Tocris Bioscience and Fisher Scientific.
The term “suitable culture media” as used herein means a culture media supportive of the particular cell type to be cultured. For example, a suitable culture media for neural progenitor cells or cells derived therefrom, such as PSC media, NEM or NIM, for example as described herein, comprising one or more additives_for example B27 or similar additive and optionally N2 or similar additive, appropriate for the stage of cells. Typically the culture media will include non essential amino acids such as Glycine, L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic Acid, L-Proline, L-Serine, glucose or equivalent, sodium pyruvate, Catalase , Glutathione reduced, Insulin , Superoxide Dismutase, Holo-Transferrin , Triiodothyronine (T3), L-carnitine , Ethanolamine, D+-galactose, Putrescine, Sodium selenite, Corticosterone, Linoleic acid, Linolenic acid, Progesterone , Retinol acetate, DL-alpha tocopherol (vit E), DL-alpha tocopherol acetate , Oleic acid, Pipecolic acid, Biotin, optionally a FGF2 agonist such as FGF2 or SUN11602, EGFR agonist such as EGF or betacelluin, and optionally heparin are added.
The term “NEM” or “neural expansion media” or variations thereof as used herein means a base media suitable for culturing neural progenitor cells such as DMEM/F12, Neuralbasal Media etc comprising one or more of sodium pyruvate, a glutamine product such as glutamine or GlutaMAX™, one or more antibiotics such as penicillin and/or streptomycin, a supplement such as B27 supplement without vitamin A or equivalent (e.g. without RA or RA analog) and N2. Agonists can be added to the NEM depending on the stage of cell differentiation, for example one or more of an FGFR agonists such as FGF2, an EGFR agonist such as EGF and/or heparin. An example of a suitable NEM is provided in Example 1. Other suitable medias, supplements, antibiotics etc are known in the art and can be used.
The term “Notch agonist” or ““Notch signaling activator” or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that binds any Notch receptor and induces proteolytic cleavage and release of the Notch receptor intracellular domain. Examples include DLL1, DLL4, Jagged1, Jagged 2, including human and other mammalian versions thereof.
The term “Wnt agonist” or “Wnt signaling activator” or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that binds and activates a Wnt receptor. Examples include AZD2858, Wnt agonist 1, CP21R7 (CP21), Wnt, or BML-284 hydrochloride. Human and other mammalian versions of the biomolecule subset thereof are contemplated.
The term “Wnt inhibitor” or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that inhibits the Wnt signalling pathway. Examples include XAV939, DKK1, DKK-2, DKK-3, Dkk-4, POCN, C59, LGK-974, SFRP-1, SFRP- 2,SFRP-5,SFRP-3,SFRP-4,WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, 1WP-L6 and derivatives thereof as well as combinations thereof.
The term “RAR agonist” or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that binds and activates a RA receptor, including for example RA or an RA analog including for example EC23.
The term “B27” or variations thereof as used herein refers to a serum free vitamin containing supplement that supports neurons and which is used with neuronal cell culture. Any such supplement that permits feeder layer independent growth can be used. B27 supplement includes for example Catalase, Glutathione, Insulin, Superoxide Dismutase, Human Holo-Transferrin, T3, L-carnitine, Ethanolamine, D+-galactose, Putrescine, Sodium selenite, Corticosterone at, Linoleic acid, Linolenic acid, Progesterone at, Retinol acetate, DL-alpha tocopherol (vit E), DL-alpha tocopherol acetate, Oleic acid, Pipecolic acid-, and Biotin.
The term “rosette” or variations thereof as used herein refers to a cellular pattern of columnar cells. The express Sox1. The neural rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells; rosettes are radial arrangements of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. In addition to similar morphology, neuroprogenitors within neural rosettes can differentiate into the main classes of progeny of neuroepithelial cells in vivo: neurons, oligodendrocytes, and astrocytes.
The term “caudalized NPCs” or variations thereof as used herein refers to NPCs having a caudal spinal cord progenitor fate and which express Sox2, Pax6 and an increased expression of Nkx6.1 relative to un-patterned NPCs and a decreased expression of Six3, DIx2, Otx2 and FoxG1 relative to un-patterned NPCs. For example, “caudalized NPCs” express Sox2, Nestin and Pax6 with equivalent level to un-patterned NPCs, and have for example at least 75% decreased level of expression for FoxG1, Otx2 and Gbx2, at least 25% increased expression Nkx6.1, and have at least 25-50% increased expression of HoxA4, HoxB4 HoxC4 and HoxC5, all relative to un-patterned-NPCs. The expression level of Nkx6.1 is for example at least 25% less than the expression level this gene compared to ventralized-NPCs.
As used herein “neural induction media” or “NIM” or variations thereof herein means a base media suitable for culturing neural precursor cells such as DM EM/F12 comprising one or more of sodium pyruvate, a glutamine product such as glutamine or GlutaMAX™, one or more antibiotics such as penicillin and/or streptomycin, a supplement such as B27 supplement without vitamin A, non-essential amino acids such as Glycine, L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic Acid, L-Proline, L-Serine, to which BMP inhibitor such as LDN193189 or Noggin, TGF8 inhibitor (such as SB431542), FGFR agonist such as FGF2, optionally heparin and EGFR agonist, optionally EGF. An example of a suitable NIM is provided in Example 1 in Table 1.
The term “pluripotent stem cell” or variations thereof as used herein refers to a cell with the capacity, under different conditions, to differentiate to more than one differentiated cell type, and for example the capacity to differentiate to cell types characteristic of the three germ cell layers, and includes embryonic stem cells and induced pluripotent stem cells, which are reprogrammed from somatic cells. Pluripotent cells are characterized by their ability to differentiate to more than one cell type using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell marker. Induced pluripotent stem cells (iPSCs) including human iPSCs (hiPSCs) can be from any somatic cell for example skin keratinocytes or fibroblasts and can be derived from a subject or a cell line. PSCs can for example be fetal derived, embryonic derived, or human embryonic stem cell derived.
The term “stem cell” or variations thereof as used herein, refers to an undifferentiated cell which is capable of proliferation, self-renewal and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells. The daughter cells can for example be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
The term “cell culture medium” or variations thereof (also referred to herein as a “culture medium” or “medium”) as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation and optionally differentiation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, vitamins etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art.
The term “passaging”, “passaged” or “passage” or variations thereof as used herein refers to transferring the cultured cells from their current growth medium to a new growth medium. Cells can be passaged for example according to as described in Example 1. Any suitable method of passaging however can be used. For example hiPSCs should be passaged in order to avoid overgrowth and to maintain them in an undifferentiated state. Further it may be preferable to passage iPSCs in clumps.
As a person skilled in the art would understand, cells can be dislodged from the culture plate with the use of enzymes and enzyme cell detachment solutions such as the enzyme cell detachment solution Accutase™. Other enzymes like Dispase, ReLeSR or TrypLE can also be used. In addition non-enzymatic methods, like EDTA solution, can also be used.
The term “N2” and non-commercial preparations thereof referred to as “hormone mix” refers to a hormone mix comprising transferrin, insulin, putrescine, selenium and prodesterone. For example N2 can comprise 10 mg/ml Transferrin, 2.5 mg/ml Insulin, 1 mg/ml Putrescine, 1 ul/ml 15 Selenium, 1 μl/ml Prodesterone. N2 can be purchased commercially from Gibco (Invitrogen/Themor scientific), Sigma and others or can be prepared.
The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.
The inventors have identified improved methods for producing spinal identity neural progenitor cells (spNPCs) from unpatterned NPCs and intermediate cell populations along the path to spNPCs.
The method can comprise one or more features of Steps 2 and 3 in Example 1. The features can be in timing, order, composition and/or selection of factors.
For example, Example 1 demonstrates that suitable culture media supplemented with FGF2, EGF and 740Y-P or a synthetic agonist of 740Y-P is useful for promoting and stabilizing the identity of spinal NPCs from caudalized-NPCs. Example 1 also demonstrates determining an effective range for 740Y-P. Cells prepared according to such methods, as indicated in Example 2, improve functional recovery when transplanted.
Accordingly, a first aspect of the invention comprises a method of producing spinal identity neural progenitor cells (spNPCs), the method comprising:
As mentioned herein, the method can be initiated using unpatterned progenitor cells or later stage cells such as posteriorized NPCs.
The inventors have identified that Wnt signaling activator is able to improve the activity of RA. For example, the inventors have identified that when the media in caudalizaiton step (e.g. where the posteriorized NPCs are treated with RAR agonist) is supplemented with for example Wnt3a, the increase in the level expression of Hox genes, in for example HoxA4 and/or HoxA5, is elevated in caudalized cells as compare to posteriorized cells and also the decrease in the level of expression of Gbx2, Otx2 or FoxG1 genes is boosted in caudalized cells as compared to posteriorized cells.
Stabilized cells have fixed identity and cannot go for example go back to other developmental identities by themselves without additional treatment. Stabilized cell will be maintained with their current identity in the proliferation or maintenance culture media. For example if they maintain their identity and gene expression profile as described herein for at least 5, at least 6 or at least 10 passages, they are considered stabilized.
A Rock inhibitor can be added after the first day in culture after each passage or a subset of passages, for example at least one, and optionally for one or more steps. It can be removed or left in for subsequent days if added on day 1. It can be removed for example by refreshing the media on day 2 after passage.
Notch inhibition is necessary for keeping cells in ectodermal fate. Retinoic acid (RA) and its analogs can push cells out of an ectodermal fate. Preparing spinal identity neural progenitor cells (spNPCs) requires both notch inhibition and RAR activation for example provided by RA. The inventors have determined that EGFL-7 can balance these required signaling pathways. It has dual function, it inhibits Notch and at the same time does not have adverse effect on RA signaling.
Another aspect of the invention comprises method of priming unpatterned NPCs to stay in an ectodermal cell fate, the method comprising:
The methods can be combined.
Accordingly another aspect of the invention comprises method of producing spNPCs from unpatterned NPCs, the method comprising:
Another aspect of the invention comprises method of producing spNPCs from induced pluripotent stem cells (iPSCs), the method comprising:
In some embodiments, the FGF2 agonist is FGF2, preferably human FGF2.
In some embodiments, the FGF8 agonist is FGF8, preferably human FGF8.
In some embodiments, the EGFR agonist is EGF.
In some embodiments, the RAR agonist is RA.
It is demonstrated in the Examples, that FGF8, EGF-L7 and/or 740Y-P in the steps described, are useful for obtaining spNPCs.
Embryoid bodies are three-dimensional aggregates of pluripotent stem cells and express pluripotent cell markers for example Klf4 and/or Oct4.
Neuroectodermal cells consists of cells derived from ectoderm. One of the prominent markers of these cells is Sox1
The FGF8 agonist, is optionally FGF8, preferably FGF8b although FGF8a, FGF8e or combinations thereof can also be used.
The inventors have found for example that starting from unpatterned NPCs which can be from any source, that the combination and order of factors, for example exposure to EGF-L7 followed by high concentration of FGF2 and/or FGF8, a short pulse of Wnt activation with RA, followed by RA alone and a step with 740-YP produces spNPCs.
Dual SMAD inhibition refers to inhibition of both BMP pathway and TGFβ pathways. This can be accomplished with “dual SMAD inhibitors” which refer to an inhibitor that inhibits both pathways or a combination of inhibitors that inhibit both the BMP pathway and TGFβ pathway, for example a BMP inhibitor and a TGFβ inhibitor. Examples for BMP inhibitors are: Noggin,
Dorsomorphin, LDN-193189, ML347, and LDN-212854 and DMH1. Others are also known for example SB431542, LDN-193189, PD169316, SB203580, LY364947, A77-01, A-83-01, GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-1008, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, K126894, SB- 203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, Dorsomorphin dihydrochloride and derivatives thereof as well as combinations thereof.
Examples for TGFβ inhibitors are: SB431532, PD169316, Galunisertib (LY2157299) or LY 3200882 as well as combinations thereof. Others are also known.
In another embodiment, the iPSCs are prepared from somatic cells from a mammal, such as a human. In an embodiment, the iPSCs are human iPSCs (hiPSC). In an embodiment, the iPSCs are prepared from a subject that has sustained a spinal injury. These cells can be used to prepare autologous spNPCs which can be used for example for autologous transplantation.
Various molecules can be substituted, for example, for BMP inhibition: LDN-193189, ML347, LDN-212854 and/or DMH1 can be used, for TGF-β. inhibition, PD 169316, Galunisertib (LY2157299) and/or LY 3200882 can be used, and for Wnt activation: KYA1797K, JW55 and/or POCN can be used.
In another embodiment, the unpatterned NPCs are incubated for about 3 days.
In another embodiment, the posteriorized NPCs are incubated for about an additional 3 days.
In another embodiment, the caudalized NPCs are incubated for about an additional 2 days.
In another embodiment, the concentration of FGF2 in the culture media used for producing posteriorized NPCs is from 20 ng/ml to about 150 ng/ml, for example, about 40 ng/mL.
Other FGF2 agonists can be used at a concentration that provides similar effect as FGF2.
In another embodiment, the concentration of FGF8 is from about 50 ng/ml to about 400 ng/ml for example about 200 ng/mL.
Other FGF8 agonists can be used at a concentration that provides similar effect as FGF8.
In another embodiment, the RA synthetic analog is about 0.1 μM EC23.
In another embodiment, the Wnt3a concentration is about 100 μg/ml. Other Wnt3a agonists can be used at a concentration that provides similar effect.
In another embodiment, the FGF2 agonist, optionally FGF2 or SUN11602, concentration in the culture media for passaging the NPCs until the identity of NPCs are stabilized is about 10 ng/ml.
In another embodiment, the EGF concentration is about 10 ng/ml.
Other EGFR agonists can be used at a concentration that provides similar effect as EGF.
In another embodiment, the 740Y-P concentration is about 1 μM.
In another embodiment, wherein the ROCK inhibitor is Y-27632.
In another embodiment, the concentration of the ROCK inhibitor is about 10 μM.
Other ROCK inhibitors can be used at a concentration that provide similar effect as Y-27632.
In another embodiment, the spNPCs passaged 3 to 10 passages.
In another embodiment, the concentration of EGF-L7 is about 10 ng/mL.
In another embodiment, the concentration of DLL4 is about 0.5 μM
Other Notch signaling activators can be used at concentrations that provide similar effect as DLL4.
Where an agonist, activator or inhibitor is a biomolecule such as a protein, mammalian and preferably human sequences are used.
In certain embodiments, the method further comprises enriching and/or isolating the desired cells.
Another aspect of the invention is a method comprises one or more steps as described in the examples.
The spNPCs can also be further differentiated, for example, for neuronal differentiation, the spNPCs can be cultured in the absence of EGF and FGF but in the presence of BDNF, GDNF, Ascorbic acid and cAMP. As mentioned in Example 1, differentiated cells showed neuronal morphology and expressed the neuronal marker β-III-tubulin (
Another aspect of the invention comprises a cell population comprising spNPCs or cells differentiated therefrom produced according to methods desbribed herein. The population of cells can be comprised in a composition, optionally in combination with a carrier, optionally a pharmaceutically acceptable carrier. In some embodiments, the population of cells are for use for transplantation in a recipient in need thereof. The pharmaceutically acceptable carrier can be a culture media or matrix, or freezing media, optionally GMP grade or sterile.
Another aspect of the invention comprises an isolated cell population of primed unpatterned NPCs comprising higher expression of Nest, Pax6 and Sox2 as compared to un-primed unpatterned NPCs. Another aspect of the invention comprises an isolated cell population of posteriorized NPCs comprising higher expression of HoxA4 and lower expression of Six3, DIx2, LmxA1, FoxG1, Gbx2, and/or Otx2 as compared to unpatterned NPCs. Another aspect of the invention comprises an isolated cell population of caudalized NPCs comprising higher expression of HoxA4 and lower expression of FoxG1, Gbx2, and Otx2 as compared to posteriorized NPCs. Another aspect of the invention comprises an isolated cell population of spinal NPCs comprising higher expression of HoxA4 and HoxA5 and less Gbx2 and Otx2 as compared to caudalized NPCs. The spNPCs described herein cannot be isolated or harvested in any quantity from tissue if at all. The methods described herein can be used to prepare for example autologous spNPCs, for example using fibroblasts or other somatic cells from a subject, such as a subject who has sustained a spinal injury, to prepare the isolated spNPCs.
In some embodiments, the higher or increased expression is at least 1 fold change (log2scale) higher/increased and/or the lower or reduced expression is at least 1 fold change (log2scale) lower/reduced.
Another aspect of the disclosure comprises an isolated cell population comprising spNPCs produced according to any of the methods described herein, optionally wherein the spNPCs are derived from a patient, optionally a spinal cord injury patient, optionally wherein the isolated cell population is for transplant, optionally autologous transplant.
Another aspect of the disclosure comprises a composition comprising any isolated cell population described herien and a pharmaceutically acceptable carrier, optionally a culture media or matrix, or freezing media, optionally GMP grade, xeno free media and/or sterile.
In some embodiments, any of the cell populations or compositions described herein are for use in treating a subject with a spinal cord injury or a neurodegenerative disease.
Another aspect of the disclosure is a method of treating a subject with a spinal cord injury or neurodegenerative disease or in the manufacture of a medicament for treating a subject with a spinal cord injury or neurodegenerative disease, the method comprising the administration of any of the isolated cell populations or the compositions described herein to treat a subject with a spinal cord injury or neurodegenerative disease or in the manufacture of a medicament for treating a subject with a spinal cord injury or neurodegenerative disease.
In some embodiments, the spinal injury is a cervical, thoracic or lumbar spinal cord injury, optionally acute or chronic.
In some embodiments, the neurodegenerative disease is multiple sclerosis (MS), Cerebral palsy (CP), Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, dementia, schizophrenia, paralysis, multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory neuropathies, epilepsy, prion disorders, Creutzfeldt-Jakob disease, Alper's disease, cerebellar/spinocerebellar degeneration, Batten disease, corticobasal degeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, and/or autism.
Another aspect of the disclosure comprises a method of generating human neural stem/progenitor cells or neural precursor cell with spinal cord identity (spNPCs) comprising the following steps:
Another aspect of the disclosure comprises a cell culture composition for use in a step of deriving the spinal NPCs in vitro from pluripotent stem cells, wherein the spinal NPCs express one or more detectable markers for Sox2, Pax6, Nestin or vimentin, and the spinal NPCs have the capacity to differentiate into cells of a neural lineage, comprising a base cell culture composition optionally wherein the base cell culture composition for each step is as provided in Table 1 and further comprising EGF-L7 agonist, optionally EGF-L7, for priming the neuroectodermal cells to stay in the ectodermal cell fate as in claim 27e, FGF2 agonist, optionally FGF2 and/or a FGF8 agonist, optionally FGF8, for posteriorizing the primed cells as in claim 27e), RAR agonist, optionally RA, or Wnt3a for caudaulizing the posteriorized cells as in claim 27g, and/or 740Y-P for inducing proliferation capacity as in claim 27h, optionally in a concentration or concentration range as described herein. In some embodiments, the base media for each step is described in Table 1.
In some embodiments, the BMP inhibitor is selected from the group consisting of Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854, DMH1 SB431542, LDN-193189, PD169316, SB203580, LY364947, A77-01, A-83-01, GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-1008, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SM16, NPC-30345, KÏ26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, Dorsomorphin dihydrochloride and derivatives thereof as well as combinations thereof, preferably selected from Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854, DMH1. In some embodiments, the BMP inhibitor is LDN193189.
In some embodiments, the dual SMAD inhibitors comprise a TGFβ inhibitor selected from the group consisting of SB431532, PD169316, Galunisertib (LY2157299) or LY 3200882 and a BMP inhibitor selected from Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854, DMH1 SB431542, LDN-193189, PD169316, SB203580, LY364947, A77-01, A-83-01, GVV788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-1008, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, KÏ26894, SB- 203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, Dorsomorphin dihydrochloride and derivatives thereof as well as combinations thereof, preferably selected from Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854, DMH1. Others are also known. In some embodiments, the TGFβ inhibitor is SB431542 or A-83-01.
In some embodiments, the Wnt inhibitor is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, POCN inhibitor, C59, LGK-974, SFRP-1, SFRP- 2,SFRP-Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, 1WP-L6 and derivatives thereof as well as combinations thereof. In other embodiments, the Wnt inhibitor is POCN. In further embodiments, the Wnt inhibitor is C59 or LGK-974.
In some embodiments, the pluripotent stem cell is a human pluripotent stem cell. In other embodiments, the human pluripotent stem cell is a human iPS cell or a human ES cell.
In some embodiments, the culture media further contains serum or a serum substitute. In other embodiments, the culture media comprises a ROCK inhibitor.
Another aspect of the disclosure comprises an isolated population of human pluripotent stem cell derived spinal neural stem/progenitor cells (spNPCs) produced according to any of the methods described herein, characterized in that the spinal neural stem cells express at least one of Nestin, Sox2, Pax6, optionally wherein the spNPCs comprise a phenotype similar to a conventional neural stem cell (NSC) and at least expression of one of the Hox genes (preferably Hox A4 or Hox A5). In some embodiments, at least one of the cultured cells expresses one of the detectable markers , HoxA4 or HoxA5 along with one or more detectable markers selected from Nestin, Sox2, and Pax6, wherein the amount of HoxA4 expression in the neural stem/progenitor cells is increased by at least 50% compared to the amount of HoxA4 expression in conventionally derived forebrain NPCs. In some embodiments, the cells are posteriorized cells and express more Hox genes, such as HoxA4 and/or HoxA5, and express less brain markers such as Gbx2, Otx2 and FoxG1 compared to unpatterned cells or fbNPCs. In some embodiments, the spNPCs differentiate to neurons, astrocytes or oligodendrocytes.
Another aspect of the disclosure comprises a composition comprising any of the isolated population of cells described herein and a carrier, optionally a pharmaceutically acceptable carrier, optionally a culture media or matrix, optionally GMP grade, xeno free media or sterile.
Another aspect of the disclosure comprises use of any of the population of spNPCs described herein or any composition described herein in the manufacture of a medicament for the treatment of a spinal cord injury of neurodegenerative disorder.
In some embodiments, the neurodegenerative disorder is multiple sclerosis, cerebral palsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, dementia, schizophrenia, paralysis, multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory neuropathies, epilepsy, prion disorders, Creutzfeldt-Jakob disease, Alper's disease, cerebellar/spinocerebellar degeneration, Batten disease, corticobasal degeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, and/or autism.
In some embodiments, the spNPCs are for transplantation into a brain or spinal cord of a patient.
Another aspect of the disclosure is a method of treating a spinal cord injury of neurodegenerative disorder, the method comprising the administration of any population of spNPCs described herein or any composition described herein to a patient in need thereof. In some embodiments, the spNPCs are for transplantation into a brain or spinal cord of a patient. In some embodiments, the neurodegenerative disorder is multiple sclerosis, cerebral palsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, dementia, schizophrenia, paralysis, multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory neuropathies, epilepsy, prion disorders, Creutzfeldt-Jakob disease, Alper's disease, cerebellar/spinocerebellar degeneration, Batten disease, corticobasal degeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, and/or autism.
Also provided is a composition comprising one or more of the cells generated using a method described herein, optionally in combination with a carrier.
As shown in the Examples, another aspect of the invention comprises a use of any cell population described herein or composition to treat a subject in need thereof, for example a subject with a spinal cord injury or neurodegenerative disease. The spinal injury may be a cervical or thoracic spinal cord injury, optionally acute or chronic. In an embodiment, the neurodegenerative disease is multiple sclerosis (MS), Cerebral palsy (CP), amyotrohic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, paralysis, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory and neuropathies.
Also included in other aspect are uses of said cells and compositions comprising said cells for transplanting and/or treating a subject in need thereof, for example for transplanting and/or treating a subject with a spinal cord injury or degeneration, for example caused by a neurodegenerative disease, optionally MS or CP.
The cells or compositions may be combined with one or more neuroprotective factors, and/or the subject administered a cell population described herein may also be administered one or more neuroprotective factors, for example GDNF, BDNF, NT3, NGF, and/or CNTF. In one embodiment, the cells administered compris spNPCs or cells differentiated therefrom. In one embodiment, spNPCs which as shown may express more pro-oligodendrogenic factors, which helps remyelination, are administered wtih while fbNPC or other cell types.
Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
The following non-limiting examples are illustrative of the present disclosure:
An example of a step-by-step protocol for the generation of hPSC-NPCs with a spinal identity (spNPCs) starting from hPSCs is provided herein. The three main steps for the procedure: 1) Generation of unpatterned NPCs or embryoid body (EB) formation and dual-SMAD inhibition 2) Priming NPCs to ectodermal cell fate, and 3) Patterning of NPCs into spNPCs (
Step 1: Generation of Unpatterned NPCs from hPSCs
Different methods of generating NPCs in vitro, including using “default pathway”22,23, or via inhibition of SMAD signaling pathway. There are protocols utilising inhibition of just BMP pathway using a BMP inhibitor, or utilizing dual-SMAD inhibitors, to inhibit both TGFβ and BMP. The NPCs that are generated with these protocols in vitro, first acquire rostral identity by default14, before they get patterned to get other identities.
We use EB culture with dual-SMAD inhibition to generate NPCs with rostral identity. These cells are referred to herein as unpatterned NPCs.
If the hPSCs are cultured on a fibroblast feeder layer, they can be further expanded in feeder-free conditions for 3-4 passages prior to induction of neural progenitors. This action acclimates the cells, improving culture quality and yield.
To generate EBs, small clumps of hPSC will be cultured on ultra-low adherent dishes
in hPSC culture media (without FGF2) and neural induction media for 7 days. During this period, hPSCs grow to cell aggregates which are called EBs.
Neuroectodermal induction begins when EBs are transferred into the Neural Induction Medium (NIM) (around day 4-5). Plating EBs on Matrigel or Geltrex in NIM promotes the transition of cells into the rosettes with a neuroectodermal lineage that are expressing Sox1. Sox2 is also in hPSCs, but Sox1 starts after cells get neuroectodermal fate.
FGF2 signaling is necessary for the polarization of rosettes. Fibroblast growth factor 2 (FGF2) is then added to guide the transition of the neuroectodermal cells into rosette structures. NEM is for transitioning NPCs to produce NPC that express Nestin, Sox2, and Pax6 (e.g. unpatterned NPCs).
1) Apply Accutase or ReLeSR (Stem Cell Technologies) as per manufacturer instructions to a healthy and homogeneous culture of hPSCs to separate them into cell clumps (consisting of 20-50 cells). Suspend the cells at a density of 1×104 clumps/mL in hPSC culture media and add 2 mL to ultra-low adherent 6-well dishes. Incubate for two days under standard culture conditions of 37° C. and 5% CO2 in a humidified incubator.
hPSC culture media (Table 1) without FGF is recommended.
Alternate methods of passaging to Accutase dissociation include using 0.5 mM EDTA in Dulbecco's PBS without MgCl2, CaCl2, or ReLeSR. ReLeSR selectively lifts only iPSC cells, leaving differentiated cells on the plate. This allows for quick and easy selection for regular iPSC culture as well.
2) On day two, gently tilt plates to allow cell clumps to collect in the bottom corner of the well and replace half of the media from the top with fresh hPSC media supplemented with BMP inhibitor Dorsomorphin (2 μM) and TGFβ inhibitor SB431542(10 μM). Repeat this process on day 4.
Dorsomorphin and SB431532 block the BMP and TGF-β signaling pathways, which has been shown to improve the efficiency of neural induction to greater than 80% of total cells14.
3) On day 5, gently tilt plates and replace the media with NIM without FGF2 (Table 1).
Cell aggregates in the form of EBs should be observed by day 5. EBs simulate the endogenous conditions under which pluripotent hPSCs transition into neuroectodermal cells.
4) On day 7, transfer the EBs in NIM with FGF2 to a standard 6 cm culture plate pre-coated for 1 hour at 37° C. with Matrigel or Geltrex. Wait 24 hours before performing microscopy to confirm that EBs have completely settled and adhered to the plates.
5) On day 8, replace half of the media with fresh NIM with FGF2, but without Dorsomorphin. Repeat this process daily until day 13 to 17 (varies based on PSC cell line), at which point the first neural rosettes will form and then neural tube-like structures should be observed. The cells in rosettes or neural tube-like structures, unlike those in the periphery, should express early neuroectodermal markers such as Pax6 and Sox1.
6) Two days after visualizing neural rosettes or neural tube-like structures, use a fine pipette tip to lift and transfer rosettes to a 15 mL Falcon tube containing NEM. Make sure to leave the non-neural cells at the periphery of the plate, as improper selection will impair NPC purity (
Alternatively one can use either Neural Rosette Selection Reagent (Stem Cell Technologies), or a brief incubation (3-5 min) with Dispase, tapping, and a PBS wash to lift neural rosettes. Neural Rosette Selection Reagent had been found to be sub-optimal for selectively lifting neural rosettes of monolayer differentiation cultures, so use in only EB cultures is recommended.
7) Resuspend the selected rosettes at 1×105 cells/mL in NEM and plate the cells onto PLL (0.1 mg/ml solution) and laminin pre-coated plates at 1×105 cells/cm2. Avoid re-plating at lower densities, as this promote undesired differentiation and loss of secondary rosette formation.
Laminin-511 (but not -332,-111, or -411) is preferred over other ECM replacements, such as Matrigel or Geltrex due to it being growth-factor free, which may interfere with the differentiation process.
After 7-8 days, lift the secondary neural rosettes manually (or with Dispase), and transfer them to another Matrigel-coated plate with N2B27 media. Following that, dissect the tertiary rosettes to purify NPCs.
Following re-plating, the culture should contain isolated NPCs that express Nestin, Pax6, and Sox2, but not Oct4.
8) Culture cells at 10 cells/μL on ultra-low adherent plates in NEM. Tilt plates and replace half the media with fresh media every 2-3 days for one week. At this stage, primary neurospheres should be observed as perfect spherical clusters of cells with a smooth contour and be at least 50 μm up to 150 μm in size. Neurospheres should not appear dark and ragged nor contain vacuoles or dead cells. They may also express Oct4, the marker for primitive NPCs.
9) Following detection, transfer neurospheres to 15 mL Falcon tubes with 500 μL of NEM. Use a flame-polished Pasteur pipette to pipette the media up and down 10-20 times, or until separation into single cells is observed. Plate the suspension at 10 cells/μL on ultra-low adherent plates in NEM.
10) Culture single cells at 1×104 cells/cm2 on PLUlaminin pre-coated standard culture plates in NEM containing 10 μM ROCK inhibitor. The next day, replace with fresh media containing NEM only.
11) After 5-6 days, use Accutase to passage cells to new PLL/laminin pre-coated plates with NEM. One day after passaging, supplement with 10 μM ROCK inhibitor.
Note: hPSC-NPCs generated using this method will, by default, express FoxG1, Gbx2 and Otx2, markers of forebrain to midbrain identity. Cells will not express HoxC4, a marker of spinal identity in NPCs.
During Step 1, Bone Morphogenetic Protein 4 (BMP4) signaling was inhibited by BMP inhibitor Dorsomorphin., LDN193189 (LDN) or Noggin can also be used, and TGFβ was inhibited by SB431542 (SB) to prevent mesodermal and endodermal differentiation. In the next step (Step 3) we are going to use Retinoic Acid (RA). RA tends to deviate differentiation of cells to a mesodermal fate26.
To keep cells in the ectodermal fate, we need to inhibit Notch signaling when RA is active27. It has been shown that inhibition of Notch signaling inhibits the differentiation to mesodermal fate and keep cells in the ectodermal layer28. To inhibit notch signaling, we use the Notch antagonist EGF-L7 (10 ng/mL). EGF-L7 interacts with all the four Notch receptors (Notch1-4) and inhibits/competes with Jagged1 and Jagged2 proteins (not DLL4) for their interaction with Notch receptors29. EGF-L7 knockdown stimulates the Notch pathway and EGF-L7 over-expression inhibits the Notch pathway. While NPCs are actively proliferating, Notch signaling contributes to the maintenance of the undifferentiated state.
Furthermore, by replacing the EGF in culture media with 10 ng/ml EGF-L7 in this step, EGF-L7 activates EGF-receptor, but it is less potent than EGF and modulates Notch signaling which reduce the hyper-proliferation of NPCs. Optionally we can also add 0.5 μM of DLL4 (DLL4: Delta-Like 4; a Notch signaling activator) with EGF-L7 to balance the reduction in Notch activity and keep the level of expression of neural progenitor genes like Nestin and Pax6 (
There is some evidence that during development, unlike anterior neural progenitors, spinal progenitors can be also originated from neuromesodermal progenitors (NMPs). NMPs are able to differentiate into both paraxial mesodermal tissue and posterior neural tissue in vitro, and even further into specific neuron subpopulations such as motor neurons30, 31. In vivo experiments in zebrafish have found that subpopulations of NMPs become fate restricted and spatially segregated, as well as having large differences in self-renewal potential32.
To generate spNPCs, we patterned cells using a stepwise treatment of morphogens33. Patterning of primed NPCs towards a spinal cord identity is modelled on the developmental cues that are involved in the formation of the spinal cord during embryogenesis.
Early in differentiation, all neural cells have a rostral identity. However, future caudal cells gradually become posteriorized and then get the characteristics of the caudal midbrain and spinal cord. FGFs, Wnt, retinoic acid (RA), and Shh are involved in this spinal cord specification and subsequent elongation. Among RA, Wnt, and FGF signals, RA causes the strongest level of caudalization: inducing suppression of forebrain differentiation and promotion of caudal CNS specification.
The region of the neural plate giving rise to the spinal cord is specified in an FGF-dependent manner. Several FGFs, including FGF3, FGF4, FGF8, FGF13, FGF18, are involved in spinal cord specification. In vitro experiments have shown that exposure of neural tissue to increasing FGF levels results in progressively elevated levels of HOXC6, HOXC8, HOXC9, or HOXC1034,35. Furthermore, several signaling pathways influence FGF8 expression. The Wnt and Shh pathways, which are active in the caudal region of the neural tube, can themselves increase FGF8 levels36,37.
1) Dissociate cells into single cells using Accutase. Plate cells at 1×104 cells/cm2 on PLL/laminin pre-coated standard culture plates in DMEM:F12 media supplemented with B27, N2, FGF2 (40 ng/ml), FGF8 (200 ng/ml). Incubate under standard conditions for three days33.
Table 2 contains a list of material that can be used for this protocol.
In this step we are using at least 2× more FGF2 (from 20 ng/ml to 150 ng/ml) and a high concentration of FGF8 (from 50 ng/ml to 400 ng/ml). In the embryo, caudal cells are exposed to select FGFs for longer periods of time than rostral cells they are involved in regionalization of the spinal cord along the rostral-caudal axis. During later stages of spinal cord elongation, FGF8 is more broadly expressed. Expression of FGF8 continues for several days but declines toward the final stages of somitogenesis and the cessation of axis elongation38, 39. Treatment with FGF8 at this concentration and time period results in posteriorization of the cells. The posteriorized NPCs produced at the end of this stage express more Hox genes, such as HoxA4, and have reduced expression of at least one of the brain markers such as Gbx2, Otx2 and FoxG1 compared to un-patterned cells (
2) On day 3, use Accutase to passage cells to new PLL/laminin pre-coated standard culture plates in DMEM:F12 media supplemented with B27, N2, 0.1 μM EC23, and Wnt3a (100 μg/ml). Incubate for an additional 3 days.
In this step we induce caudalization of cells using retinoic acid (RA) or the synthetic retinoid analog, EC23. Using EC23 is preferred as it is more photostable at incubation temperatures.
FGF and RA signaling are not sufficient (alone or together) to induce caudal characteristics in neural cells grown in vitro and Wnt signaling (Wnt3a) is further required to specify neural cells to a caudal identity42.
3) On day 6, use Accutase to passage cells to new PLL/laminin pre-coated standard culture plates in DMEM:F12 media supplemented with B27, N2 and 0.1 μM EC23. Incubate for an additional 2 days.
No Wnt3a is required at this stage.
Treatment with RA and Wnt for 3 days results in caudalization of cells. These caudalized NPCs express Hox genes such as HoxA4. EC23 is continued for an additional 2 days after passaging to stabilize the caudal identity of the NPCs. This additional RA pathway activation results in a significant reduction (to nearly no expression) of Gbx2, Otx2 and FoxG1 levels compared to posteriorized cells (
4) Passage Caudalized-NPCs for 2-3 passages in DMEM:F12 supplemented with B27, N2, FGF2 (10 ng/ml), EGF (10 ng/ml) and 740Y-P (1 μM) until the identity of cells get stabilized, at this stage spinal NPCs are formed. Spinal NPCs can be passed and maintained in this media for up 3-5 more passages for optimum results, but passing up to P10 (and beyond) can be acceptable depending on the culture conditions (
during maintenance period, the proliferation rate of cells is reduced. To generate
sufficient numbers of cells, prolonged culture for several passages is required. The concentration of FGF2 cannot be increased at this stage. To overcome this problem, 740Y-P is added which is as effective as FGF2 at promoting neuronal cell survival and proliferation via PI 3-kinase-Akt pathway43. The effect of 740Y-P is dose dependent.
We recommend using spNPCs between ˜P3-P10. However, later passage cells may develop NPCs with mixed identity and cells that generate more GABA-ergic interneurons
After each passage, add 10 μM Rock inhibitor (Y-27632) on day 1 of culture.
The self-renewal capability spNPCs is comparable to that of fbNPCs, as it was determined using a clonal analysis (
The developmental potential of spNPCs are comparablee to that of fbNPCs as assesses by analysing their capacity for differentiation into the three main neural lineages. For neuronal differentiation, NPCs were cultured in the absence of EGF and FGF but in the presence of BDNF, GDNF, Ascorbic acid and cAMP. Differentiated cells showed neuronal morphology and expressed the neuronal marker β-III-tubulin (
The spontaneous synaptic activity of neurons derived from spNPCs is comparable to that of fbNPCs as determined using whole-cell patch-clamp recordings that demonstrated both lines had similar inward sodium currents and were able to generate action potentials. Furthermore, the amplitude of sodium currents and the firing properties of neurons were not significantly different between the two groups (
Multifaceted insults to the spinal cord after trauma or due to degenerative conditions can induce lasting sensorimotor deficits which hinder a patient's function. These impairments are largely due to tissue loss making targeted cell replacement a promising therapeutic strategy44. The successful generation of spNPCs from iPSCs marks a significant milestone in developing effective cell-based treatments for patients where none currently exist. With the unique ability to match the niche of the target tissue, spNPCs have demonstrated improved integration and survival when transplanted into the spinal cord15,16 Of particular interest is their ability to regrow the CST and V2a circuit interneurons, which are crucial in restoring lost motor function15,16. In contrast, conventional brain NPCs differentiate poorly into these populations and also produce undesired cortical cells which hinders their potential to produce meaningful outcomes. Together, these findings underscore the importance of matching the cell identity to the affected region to maximize regrowth and recovery.
The shift from forebrain-NPCs to spNPCs marks a key step in the optimization of cell-based therapies for degenerative spinal conditions.
Neurodegeneration and the subsequent lack of regeneration pose a major obstacle to the functional recovery of spine patients. Stem cell therapy is poised to remove this obstacle, with recent advances in cell programming moving the treatment one step closer to clinical application. Specifically, assigning iPSC-NPCs with spinal identity allows improved integration, repopulating the site of damage with the appropriate cells without introducing foreign cells originating from outside the spinal cord.
This study seeks to determine how the differentiation state of transplanted neural stem/progenitor cells is modulated by the spinal microenvironment to promote recovery. Using a rodent model of cervical spinal cord injury, NPCs at different stages of development (fbNPC vs spNPCs) were transplanted into a rodent model of cervical spinal cord injury and the mechanism(s) of recovery were examined.
Transplantation of tripotent neural stem/progenitor cells is a promising therapeutic strategy for traumatic spinal cord injury (SCI), however, the optimal temporal and spatial developmental stage for transplanted cells remains to be determined. In this study, we compared the fate determination of neuroepithelial stem/progenitor cells (NPCs) with an anterior forebrain identity (fbNPC) to NPCs patterned to acquire a ventral spinal cord identity (spNPCs) in the injured spinal cord microenvironment both makes Glutamatergic or GABAergic interneurons. Human induced Pluripotent Stem Cell (hiPSC) derived fbNPC and spNPCs were generated and transplanted into the injured spinal cord. fbNPC mainly differentiated into neurons, while spNPCs mainly differentiated to myelinating oligodendrocytes. The unique differentiation profiles were mainly due to differential Pax6 expression between the two lines, and were affected by activation of Notch signaling in the injured spinal cord microenvironment. Transplantation of both NPCNPCS lines resulted in neurobehavioral recovery, including improvements in forelimb grip strength and measures of forelimb/hindlimb locomotion, as assessed by Catwalk. fbNPC produced their effects in functional recovery through differentiation to neurons, which migrated towards the cavity and formed a cellular bridge. However, spNPCs produced their effects through remyelination. Both lines provided trophic support for tissue preservation and regeneration.
Characterization of in vitro Generated Cells
GFP-positive-hiPSCs, generated by non-viral, piggyBac transposon-mediated reprogramming (Hussein et al., 2011), were used to establish fbNPC and spNPCs by mimicking key morphogenic cues and replicating developmental neural tube patterning in vitro (
A comparative gene expression analysis revealed that the expression of pluripotent cell markers (Oct4, Nanog) was decreased, whereas expression of neural cell markers (Sox2, Pax6 and Nestin) was increased in both lines compared to the original hiPSCs. The expression of Nestin and Sox2 was comparable between fbNPC and spNPCs (
The Injured Spinal Cord Microenvironment has a Distinct Effect on the Proliferation and Differentiation of fbNPC and spNPCs
During CNS development, neurons, astrocytes and oligodendrocytes arise from common neuro epithelial progenitor cells in a process guided by the dynamic interplay of environmental signals(Silbereis et al., 2016). However, after SCI, these developmental cues are not present and different environmental factors are expressed.
This difference in the composition of differentiation factors between the injured and uninjured spinal cord microenvironment could have distinct effects on the proliferation and fate determination of NPCs that are from different developmental stages. To assess this effect, we exposed fbNPC and spNPCs in vitro to naïve spinal cord homogenate (Naïve-h) or injured homogenate (SCI-h). These homogenates were prepared from uninjured cervical spinal cord tissue or cervical injured tissue two weeks post injury.
To investigate the effect of Naïve-h or SCI-h on the self-renewal ability of NPCs at different developmental stages, a clonal analysis was performed for fbNPC and spNPCs. Neurospheres were mechanically dissociated to a single cell suspension and plated at clonal density in the neurosphere assay (
To explore the effect of factors present in the microenvironment of naïve or injured spinal cords on the differentiation of NPC lines, we differentiated cells in the presence of Naïve-h or SCI-h. Culturing NPC lines in the presence of Naïve-h for 4 weeks resulted in their differentiation to neurons (βIII-tubulin+), astrocytes (GFAP+) and oligodendrocytes (O1+), confirming their tripotency. In Naïve-h, fbNPC differentiated to more neurons (31.7±2.0%,) compared to spNPCs (20.5±1.9%, p<0.5), while spNPCs differentiated to more O1 expressing oligodendrocyte (fbNPC; 31.7±2.0% v.s. spNPCs; 20.5±1.9%, p<0.5). However, culturing in the presence of SCI-h had a distinct effect on fbNPC compared to spNPCs. A larger portion of fbNPC remained undifferentiated (Nestin+; 27.7±3.5%) or differentiated to neurons (29.4±2.8%), while spNPCs mainly differentiated along the astroglial and oligodendroglial lineage (O1+; 37.8±5.3, GFAP+34.0±3.1). Furthermore, a lower percentage of spNPCs remained undifferentiated (Nestin+; 18.29±3.8%) (
Divergent Effects of Notch Signaling on fbNPC and spNPCs
Our results showed that SCI-h induces proliferation of NPCs when they are at an early developmental stage (fbNPC), while inducing their differentiation to an astroglial lineage when they are at a later developmental stage (spNPCs). This finding suggests that there are factors in the SCI niche which have different effects on early vs. late developmental stage NPCs.
Notch signaling is a pathway involved in binary cell fate decisions as well as induction or enhancement of terminal differentiation. During early CNS development, Notch signaling is first used to induce the self-renewal and proliferation of cells. However late in development, Notch induces the differentiation of cells to a glial fate(Grandbarbe et al., 2003; Namihira et al., 2009; Tanigaki et al., 2001). Therefore, Notch signaling is a potential candidate responsible for the observed developmental stage differences in differentiation after exposure to SCI-h. We analyzed the expression of Notch activating ligand, DLL1, in Naïve-h and SCI-h using western blotting and found that the expression of DLL1 was highly upregulated after SCI (
The Pax6-regulated activity in neural stem cells is highly dose sensitive, with increasing Pax6 levels driving the system towards neurogenesis. Relative levels of Pax6 and Notch signaling are factors in a dynamic balance that controls whether neural stem cells self-renew, differentiate to neurons, or generate glial progenitor cells. In the presence of Notch signaling, increased Pax6 activity suppresses neurogenesis and promotes self-renewal, in part by repression of Neurog2. Conversely, decreased levels of Pax6 in the presence of Notch signaling result in gliogenesis.
fbNPC and spNPCs Survived, Migrated and Differentiated in the Injured Spinal Cord
To investigate the in vivo behavior of fbNPC and spNPCs after transplantation, T-cell deficient RNU rats received a C6/7 cervical SCI followed by cell transplantation at 2 weeks post-injury. Transplanted cells (GFP+) were found in both the white and gray matter. Grafted fbNPC were located mainly around the lesion epicenter (
The chemotactic response of neural stem cells to the injury site is highly related to their differentiation status and the type of lesion(Filippo et al., 2013; Imitola et al., 2004). Chemokines, such as CXCL12, are expressed within the SCI epicenter and attract neural progenitors that express CXCL12 receptors, CXCR4 or CXCR7(Chen et al., 2015; Imitola et al., 2004). fbNPC express higher levels of chemoattractant receptors CXCR4 and CXCR7, cell adhesion molecules (e.g. CD44) and integrins (e.g. ITGA4), as compared to spNPCs, which may explain this chemotactic response (
Quantification of GFP+ cells revealed extensive survival of transplanted cells within the injured spinal cord (
In terms of differentiation, both fbNPC and spNPCs differentiated into neurons, astrocytes, and oligodendrocytes in vivo, however, a proportion of cells in both lines remained in an undifferentiated Nestin positive state. The number of nestin positive cells for the fbNPC line was more than two times greater than spNPCs (fbNPC : 31.2±7.1 and spNPCs : 13.21±4.5%) (
We investigated the expression of Ki67 in GFP+grafted cells to identify
hyperproliferative or immature grafted cells (
In order to be fully functional, the neurons derived from transplanted cells need to be myelinated. In addition to myelination, graft derived oligodendrocytes can contribute to the remyelination of denuded axons. To explore this, we performed immunohistological analyses using myelin basic protein (MBP) as well as NF200. Few GFP+ /MBP+ cells were observed in the fbNPC group, while numerous GFP+/MBP+ double-positive cells were observed in the spNPC group, suggesting that transplanted spNPCs differentiated into myelinating oligodendrocytes (
fbNPC and spNPC Transplantation Enhances Tissue Preservation After SCI
One of the main beneficial effects of transplanted NPCs is the secretion of trophic factors. Theses trophic factors have pro-neurogenic, pro-axonogenic, as well as various anti-apoptotic and pro-angiogenic effects, which preserve endogenous tissue(Ruff et al., 2012). To address the trophic factors which are secreted by fbNPC and spNPCs, we used a human growth factor antibody array to analyze the secretion of different growth factors between fbNPC and spNPCs. Both lines showed expression of different fibroblast growth factor (FGF) isoforms at an increased level. FGF isoforms have been linked to survival and neurite outgrowth in certain neuronal subtypes(Pataky et al., 2000). Also, pro-neurogenic trophic factors, such as NT3, NGF, and GDNF, were expressed by both lines at a high level. Interestingly, trophic factors that induce differentiation and proliferation of glial lineages, like PDGF and TGF isoforms, were increasingly expressed by spNPCs, while pro-angiogenic factors like VEGF were greatly expressed by fbNPC (
Secretion of these trophic factors in vivo can enhance endogenous tissue preservation. Our histomorphometric analyses using LFB and H/E staining demonstrated that more white matter was present in spNPC treated animals as compared to animals treated with vehicle, but only at 240 and 480 μm caudal to the epicenter (
In addition to this histomorphometric analysis, we further investigated cavitation and functional vascularity by using high resolution ultrasound (VHRUS) imaging(Soubeyrand et al., 2014). VHRUS imaging is capable of generating planar full-depth images and 3D reconstruction volumes of cavitation in situ. This helps to overcome difficulties with tissue shrinkage that occur during routine histological processing(Soubeyrand et al., 2014). The cavity size under VHRUS was significantly smaller in fbNPC and spNPC transplanted animals compared with vehicle treated animals (vehicle: 2.79±2.55 mm3, spNPCs: 1.37±0.25 mm3; p<0.05, fbNPC: 0.15±0.05 mm3; p<0.005, n=5,
spNPC-Derived Neurons Make Synaptic Connections with Endogenous Cells and Enhance Electric Conduction
The neurons that are differentiated from transplanted cells must form synaptic connections with endogenous cells and integrate into local networks to promote functional recovery. Using immunostaining with Synapsin I (SYN1) antibody and immunotransmission electron microscopy, we assessed whether gold-labeled GFP+ cells formed synaptic connections with label-negative endogenous cells (
Transplantation of fbNPC and spNPCs Improves Functional Recovery
Next, we investigated whether transplantation of fbNPC and spNPCs was associated with functional recovery. Forelimb strength and trunk stability were assessed with grip strength and inclined plane behavioral tasks(Wilcox et al., 2017). All injured animals consistently recovered forelimb grip strength over the assessment period, although recovery trajectories diverged at approximately 4 weeks post-transplantation. There was a significant improvement in forelimb grip strength in both fbNPC and spNPC groups compared to the vehicle control group (p<0.05) (
Increased neuropathic pain after cell-based treatment is a potential concern(Hofstetter et al., 2005). Hence, we assessed thermal and mechanical allodynia (experimental procedure in supplemental information). Latency times for the rat to remove its tail from a focal heat source (
We have chosen to compare cells with anterior brain vs. ventral spinal cord identity because these cells demonstrated lower expression of Ptf1a (
We demonstrated that the expression of BMPs, TFGβ, Jagged1/2 and Notch protein are upregulated in our model of RNU rat cervical SCI 2 weeks after injury relative to the uninjured (naïve) cervical spinal cord. When we culture the cells in baseline differentiation medium, the number of cells differentiating to O1+ cells was not significantly different for spNPCs as compared to fbNPCs (
However, to have a global understanding of the gene expression profile for fbNPCs and spNPCs, we performed RNAseq analysis between the two lines. We demonstrated that fbNPCs express higher levels of chemoattractant receptors CXCR4 and CXCR7, cell adhesion molecules (e.g. CD44) and integrins (e.g. ITGA4), as compared to spNPCs, which may explain this chemotactic response. (
Understanding various mechanisms involved in the obtained functional recovery is important and has implications for the design of regenerative therapies for SCI. In general, trophic support has a faster effect on functional recovery, therefore the functional recovery that is emerged in the first 2-3 weeks after transplantation, is most probably attributed to trophic support. It takes more than 3 weeks for human cells to differentiate to functionally active neurons or myelinating cells7. Therefore, the portion of the recovery that is emerged after this time, can have an additive effect of both trophic support and cell integration (
The optimum temporospatial developmental stage of neural stem/progenitor cells must be determined in order to produce cell transplantation strategies with the highest likelihood of improving outcomes post-SCI. In the current study, we compared the therapeutic potential of hiPSC-derived neuroepithelial stem progenitor cells at two distinct stages of their development in a cervical model of SCI. fbNPC represent a very early stage of development of neural progenitor cells, which have anterior identity, while spNPCs demonstrate a later stage of development of neural progenitors with a caudal ventral identity. Our results demonstrated that both cell types contributed to functional recovery, but through different mechanisms.
Understanding the reciprocal interaction between the SCI microenvironment and cells with different identities will allow the field to more closely target the graft to a patient's requirements. The injured spinal cord microenvironment showed distinct effects on the proliferation and differentiation of fbNPC and spNPCs. The self-renewal potential of fbNPC increased upon exposure to the SCI niche, while this environment biased the differentiation of spNPCs towards a glial linage. This contradictory effect is a typical hallmark of Notch signaling on primitive vs. definitive neural progenitors (Grandbarbe et al., 2003; Namihira et al., 2009; Salewski et al., 2013; Tanigaki et al., 2001). This is in concordance with activation of Notch signaling in the SCI microenvironment (
Here, we demonstrate that the interplay between graft and host tissue is two-sided. Transplanted NPCs produced effects on the injured tissue through both cell-autonomous (cell replacement) and noncell-autonomous (trophic) mechanisms. Both NPC lines differentiated to neurons, oligodendrocytes and astrocytes, although in different proportions. Differentiated fbNPC mainly consisted of neurons, while spNPCs predominantly differentiated to oligodendrocytes. Importantly, the NPC derived neurons could potentially form synapses with endogenous neurons and improve neural circuit conduction(Lu et al., 2012, 2014) by bridging and relaying supraspinal neurons from the cortex to their spinal targets(Bonner and Steward, 2015).
The NPC derived oligodendrocytes could potentially contribute to remyelination. spNPCs mainly differentiated to Olig2 and/or APC positive oligodendrocytes. These cells could differentiate to MBP positive myelinating oligodendrocytes (
After SCI, cystic cavities form as a result of cell death and the clearance of damaged tissue by phagocytotic cells(Dusart and Schwab, 1994; Liu et al., 1997). This damage to the spinal cord significantly affects functional recovery. Other than replacing lost cells, transplanted NPCs (mainly fbNPC) contributed to forming a cellular bridge through the cystic cavity. This cellular bridge provides a structural substratum that allows injured axons to grow into a permissive environment.
Migration towards the cavity (pathotropism) is very important for forming this cellular bridge. However, the chemotactic responses of neural stem cells to the injury site is related to their differentiation status and expression of receptors for chemoattractants (Filippo et al., 2013; Imitola et al., 2004). Chemoattractants, like CXCL12 (SDF1), are expressed by the injured site post-SCI, and attract cells that express CXCL12 receptors, CXCR4 or CXCR7(Chen et al., 2015; Imitola et al., 2004). It has been shown that neural stem/progenitor cells at their early stages of development show higher expression of these receptors and a higher degree of pathotropism (Chang et al., 2013; Ferrari et al., 2012). This is in concordance with our results showing that early stage NPCs (fbNPC) express higher levels of CXCR4 and CXCR7 than spNCEs, and demonstrate a higher tendency to migrate towards the cavity and form a cellular bridge.
In addition to cell replacement, transplanted NPCs provide trophic support to the injured tissue. Secretion of trophic factors promotes tissue repair by preventing tissue damage and enhancing the survival of endogenous neural cells. It also re-establishes the functional interactions between neural and glial cells(Ruff et al., 2012). Both NPC lines secreted elevated levels of trophic factors like FGF, NT3, NGF, and GDNF, which are involved in the survival and regeneration of injured tissue. The composition of secreted growth factors was slightly different between the cell types, with spNPCs expressing more pro-oligodendrogenic factors, which helps remyelination, while fbNPC expressed more pro-angiogenic factors that help restore vascularity in the injured tissue.
As research continues into strategies to generate neural stem/progenitor cells with different identities, recognizing the optimal differentiation state of cells for transplantation to treat SCI is vital for clinical translation. Overall, our study demonstrates that transplantation of human fbNPC and spNPCs can exert positive effects through trophic support and cell replacement. Both fbNPC and spNPCs secrete a plethora of different trophic factors. A proportion of the functional recovery observed in fbNPC transplanted animals could be attributable to providing a permissive cellular bridge in the cavity as well as integration and connection of fbNPC derived neurons into the host neural network. Conversely, a large proportion of spNPCs' effects appear to be the result of enhanced remyelination. Both mechanisms are important for functional recovery. Further experiments are required to determine the synergistic effect of transplanting a combination of both fbNPC and spNPCs.
Generation and Characterization of fbNPC and spNPCs
Human iPSCs (hiPSCs) line PB1-53, as described before(Hussein et al., 2011), were differentiated to NPCs using dual SMAD inhibition in monolayer culture with some modifications(Varga et al.). fbNPC were maintained in DMEM:F12 supplemented with N2, B27-VA media with FGF2 (10 ng/ml) and a combination of inhibitors targeting TGFβ (SB431542) and WNT signaling (CHIR99021)(Payne et al., 2018; Varga et al.). For spNPCs, we patterned cells with stepwise treatment with patterning factors (Lippmann et al., 2015). At the first step cells were cultured in DMEM:F12 media supplemented with B27, N2, FGF2 (20 ng/ml), FGF8 (200 ng/ml) for three days(Lippmann et al., 2015). Cells were then caudalized by supplementing the culture with 0.1 μM retinoic acid agonist EC23 for an additional 5 days. Cells underwent ventralization by treatment with 1 μM sonic hedgehog (Shh) agonist Purmorphamine for 3 days. At this stage cells showed a ventral spinal cord identity. spNPCs were maintained in media consisting of B27, N2, FGF2 (10 ng/ml), EGF (10 ng/ml) and 740Y-P (1 μM) for three passages prior to transplantation. During passaging, 10 μM Rock inhibitor (Y-27632) was added on day 1. Further details are provided in Example 1.
mRNA Expression Profiling
Quantitative RT-PCR was used to examine the expression profile of fbNPC and spNPCs. mRNA was isolated using the RNeasy Mini Kit (Qiagen74104). A Nanodrop spectraphotometer was used to evaluate the concentration and purity of the mRNA. cDNA was synthesized using a SensiFAST™ cDNA Synthesis Kit (Bioline 65053) with random hexamere primers. RT-PCR was performed using TaqMan design primers with SensiFAST Probe Hi-ROX master mix (Bioline 82020) under recommended thermocycling parameters on a 7900HT Real time PCR system. Samples were run in triplicate. Values were normalized to the GAPDH housekeeping gene. For the examination of the gene expression levels, results were normalized to GAPDH and to the reference cells. The gene expression level was calculated using the 2-ΔΔCT method.
Two weeks after injury, rats were perfused with ice-cold PBS, and a 5 mm long segment of the injured spinal cord centered on the injury epicenter was removed. For naïve rats, the same region of the spinal cord as the injured rats was removed for this preparation. Spinal cords of 5 rats for each group were pooled together in 1 ml DM EM:F12. The tissue was homogenized by using a small conical mortar and pestle for 2 min, while kept on ice. The homogenate was cleared by centrifugation at 12000 g for 15 min, and the total protein concentration of cleared supernatants was measured using a BCA test. After adjusting the total protein concentration, the aliquots were kept at −80° C. until use.
In vitro Treatment with Spinal Cord Homogenate
In order to study the effect of injury induced factors on the differentiation potential of NPC lines in vitro, NPCs were cultured on their maintenance media without growth factors (no FGF2 for fbNPC and no FGF2/EGF for spNPC) and treated with 100 μg/ml cleared homogenate from the injured (SCI-h) or naive (Naive-h) spinal cord for 30 days. Cells were then fixed for 20 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) and 40% sucrose at room temperature. Following fixation, cells were permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate in PBS for min (except for O1 staining) and then placed in blocking buffer (5% BSA) for 1 h. Primary antibodies were diluted in a blocking buffer solution and incubated with the cells overnight at 4° C. Following extensive washing, samples were incubated with fluorophore-conjugated secondary antibodies for 1 h.
To explore the difference in trophic factors secreted by fbNPC and spNPCs, a membrane-based antibody array (Abcam, #ab134002) was used to compare the expression of 41 growth factors in conditioned medium prepared from cells. fbNPC and spNPCs were seeded at an equivalent density of 1×106 cells on 10 cm laminin coated plates, and after 18 h the conditioned media were collected. The conditioned media was incubated with the antibody array and developed with biotin-conjugate antibodies and HRP-Streptavidin, as per the manufacturer's instructions.
fbNPC and spNPCs were plated in their respective maintenance media w/wo Naïve-h or SCI-h (100 μg/ml) at a clonal density of 10 cells/μL in a final volume of 500 μl medium in uncoated 24-well plates (Nunc, Rochester, N.Y.). Neurospheres >50 μm in diameter were quantified after 7 days of undisturbed culture. Just prior to imaging, the content of each well was transferred to a Matrigel coated dish, incubated for 30 min and fixed with 4% PFA.
fbNPC and spNPCs were plated on laminin coated 96-well tissue culture plates (removable strip plates, Corning) at a density of 1×103 cells/100 μl/well and the cell number was determined at 12, 24, 48 and 72 hours after plating using a BrdU cell proliferation assay (Abcam, #ab126556), as recommended by the manufacturer.
All animal experiments were approved by the Animal Care Committee, University Health Network (Toronto, Ontario, Canada) in accordance with the policies of the Canadian Council of Animal Care for the use of experimental animals and under the supervision of clinical veterinarians. Adult Rowett Nude (ATN) rats 180-230 g (Charles River Laboratories) were used for cell transplantation.
The clip compression model of cervical SCI has been characterized extensively in our laboratory and described previously(Wilcox et al., 2014). Briefly, ATN rats were anesthetized using 4% isoflurane and administered 0.05 mg/kg buprenorphine and 10 ml saline, and were sedated for the remainder of the surgery under 2% isoflurane. The rats received a C6 and C7 laminectomy. A modified aneurysm clip calibrated at 21.5 g (Walsh, Oakville, Ontario, Canada) was applied extradurally to the spinal cord at the C6 vertebral level for 60 seconds and then removed. For the sham group, only a laminectomy was performed without clip compression. Gel foam (Ferrosan, Denmark) was placed on the spinal cord at the end of the surgical procedure and the incision was closed in layers using standard silk sutures. Animals were allowed to recover in their cage under a heat-lamp and, subsequently, were housed in a 12-hour light-dark cycle at 26° C. with free access to food and water. Animals received extensive postoperative care, including Clavamox in drinking water 3 days before injury until the study endpoint. Animals were administered buprenorphine (0.1 mg/kg) for 3 days and meloxicam (1.0 mg/kg) for 3 days. Injured rats were administered fluids and nutritional support, and their bladders were manually voided three times daily for 14 days as needed.
Animals were randomly divided into three groups to receive transplantation of 4×105 fbNPC, spNPCs or a vehicle (control) injection, on day 14 post-injury. Under isoflurane (1-2%) and a 1:1 mixture of O2/N2O, rats were placed in a stereotactic frame and administered 0.05 mg/kg buprenorphine and 10 ml saline, and injuries were carefully re-exposed. Cells were made ready for transplantation by treating the monolayer of both NPC lines with Accutase (0.05 ml/cm2 cell culture area) and were incubated at 37° C. for 2 min. The Accutase was then neutralized by media, cells were detached and spun at 400 g for 4 mins, re-suspended in culture media and viable cells counted. Cells were transplanted intraspinally at a density of 50,000 cells/μl. 2 μl of cell suspension were injected per site and there were four injection sites per rat (1.0 mm bilateral to the midline at 2 mm rostral and caudal). Injections were delivered at 0.6 μl/minute, left to dwell for 2 minutes, and retracted over 2 additional minutes using a Hamilton syringe and a stereotaxic injection system (System UMP3 with Micro4, World Precision Instruments, Sarasota, Fla.). The control animals also received the same number of injections to the spinal cord with only culture media.
At 10 weeks post-injury, rats were deeply anesthetized with isofluorane and transcardially perfused with 180 ml of ice-cold phosphate-buffered saline (PBS) and 180 ml of 4% paraformaldehyde in PBS. Following collection, spinal cord tissue was fixed for 5 hours and then cryoprotected in 30% sucrose in PBS for 24 hours. Spinal cord segments were embedded, frozen and stored at −80° C. Cryostat sections were made 30 μm thick along a 1.5 cm length portion of the spinal cord, and centered rostrocaudally at the site of injury.
Serial sections were stained with Luxol Fast Blue (LFB) and Hematoxylin & Eosin (H&E). LFB is a myelin-selective stain, while H&E are stains for all cell nuclei and cytoplasmic proteins, respectively(Nguyen et al., 2012). Slides were retrieved from −80° C., baked at 56° C. for 15 minutes and washed in various medium to prepare for overnight LFB staining at 56° C. The following day, tissue was retrieved from the oven and processed through various washing steps for H&E staining. Afterwards, the tissue was sequentially dehydrated with increasing concentrations of alcohol, xylene and cover slipped.
A blinded investigator performed LFB and H&E analyses on tissue±2,440 μm centered at the injury epicenter. Unbiased measurements were made using a Cavalieri volume probe from Stereo Investigator (MBF, Bioscience, Wilson, Vt.) to produce area and volume estimations of preserved white matter and lesional tissue(Wilcox et al., 2014). Lesional tissue was defined as areas with the following aberrant histology; small round cysts, irregularly shaped vacuoles, disorganization of both white and gray matter and eosinophilic neurons. Calculations and analyses were done for tissue sections every 240 μm.
In vivo Very High Resolution Ultrasound (VHRUS) Imaging
VHRUS and Power Doppler imaging were performed as previously described(Soubeyrand et al., 2014). Under isoflurane anesthesia, animals were placed within a custom-made stabilization frame on the imaging platform (Vevo imaging station, Visualsonics, Toronto, Canada). The injury was re-exposed through a mid-line incision and ultrasound gel (Scanning Gel, Medi-Inn, Canada) was placed on the dura mater. The spinal cord was scanned with the VHRUS probe (44 MHz, Vevo 770, Visualsonics, Toronto, Canada). The 3D B-mode scans were analyzed using ImageJ software and the TrakEM2 plugin to generate a reproducible cavity volume.
For Power Doppler analysis, a field-of-view with fixed area was defined and centered manually on the central sagittal slice. The Doppler signal was binarized through image thresholding, and batch analysis was carried out for all sagittal sections. The area fraction of positive Doppler signal was multiplied to the actual image area to yield a Doppler-positive area per sagittal slice, which was ultimately summed to yield the total Doppler area for each spinal cord (termed “functional vascularity”). For clarity, all values have been normalized to the sham injury Doppler signal.
Immunohistochemistry was performed as previously described(Wilcox et al., 2014). The cryostat sections were first incubated in PBS for 5 min, prior to 1 h incubation in blocking buffer (1% BSA, 5% Skim milk, and 0.3% Triton-X 100 in PBS) in RT. After removing the blocking solution, primary antibodies were incubated overnight at 4° C., followed by washing three times with PBS for 10 min (Table S1). Slides were rinsed and incubated in appropriate fluorescent secondary antibodies (all 1:500, Thermo Fisher Scientific) for 2 h at room temperature. The images were taken using a Zeiss LSM 510 or LSM880 confocal microscopes.
Frozen sections were incubated with anti-GFP mouse monoclonal antibody, and then incubated with nanogold-conjugated anti-mouse IgG secondary antibody (1:100 Invitrogen). Sections were fixed with 2.5% Glutaraldehyde, post-fixed with 0.5% OsO4 and embedded into Epon. Ultrathin sections (70 nm thick) were prepared, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (TEM, JEOL 1400 plus). For the analysis of the synaptic density and the ratio of asymmetrical/symmetrical synapses, sets of micrographs for 4 rats in the control-hiPSC-NPC group and 4 rats in the GDNF-hiPSC-NPC group were used. The synaptic counts were expressed as the number of synapses on a membrane length unit of 100 μm. The estimate of synaptic parameters was derived from 145 synapses in hiPSC-NPC and 185 synapses in the GDNF-hiPSC-NPC group. By using well-established criteria for symmetric and asymmetric synapses (Gray, 1959), we counted the number of each type of synapse on electron microscopy images.
Recording in vivo Compound Action Potentials
Electrophysiological recordings were carried out under isoflurane anesthesia (1.0-1.5% inspiratory concentration). Twisted bipolar stimulation electrodes were made with polyimide-insulated stainless-steel wires with outer diameter of 0.2 mm and electrode-tip spacing of 0.1 mm (Plastics One, Roanoke, Va., USA). An aCSF-filled glass microelectrode with a 100˜120 μm tip diameter was used for recordings. Stimulation was applied at spinal cord segment T1 at 1.2 mm depth, targeting the dorsal corticospinal tract (dCST). Stimulation protocol consisted of delivering a cathodic rectangular wave with a pulse width of 0.1 ms and an amplitude of 1.5 mA, every 10 seconds. Stimulation pulses were generated using a PSIU6 stimulus isolation unit Grass S88 stimulator (Grass Technologies, USA). Evoked compound action potentials (CAPs) were recorded from spinal cord segment C4 at 1.2 mm depth, also targeting the dCST (Li et al., 2016). The spacing between the stimulating and recording electrodes was 10 mm. CAPs were recorded in DC mode with Axoprobe 1A amplifier (Molecular Devices, CA, USA) and processed using pClamp8 software and Digidata 1320A (Molecular Devices, CA, USA) with the sampling rate of 83.33 kHz. 2 kHz low-pass and 100
Hz high pass filters were used for these recordings. The data analysis was performed offline using a custom written program in Matlab (Math Works, Natick, Mass., USA) to measure the peak-amplitude and peak-latency of the evoked CAP responses in each animal. CAP conduction velocity was calculated by dividing the electrodes' spacing (10 mm) by the measured peak-latency.
Rats from each experimental group underwent weekly neurobehavioural testing to assess forelimb strength, digital dexterity, trunk stability and function. Two independent, blinded observers measured each parameter. Forelimb strength was assessed using a force meter to measure grip strength(Garcia-Alias et al., 2009). Trunk and forelimb strength were also assessed using the inclined plane test(Bresnahan et al., 1987), which required rats to hold their standing position while the angle of the surface beneath them was incrementally increased. The test finished when the rats were no longer able to hold their position for 5 seconds. The last angle that rats were able to hold for 5 seconds was recorded. Forelimb gait analysis was performed using the Catwalk gait assessment system (Noldus Information Technology, Wageningen, Netherlands)(Dai et al., 2011),(Miyagi et al., 2011).
Results are stated as mean±standard error of the mean (SEM) or Standard deviation (SD) as indicated in the figure legends. Neurosphere assay and immunohistological data were analyzed using Student's t-tests. Histomorphometric and behavioral data were analyzed using two-way analysis of variance (ANOVA) with Tukey's post hoc test, or one-way ANOVA with Turkey's post hoc test as indicated in figure legends. The significance level of all analyses was set at p<0.05. Data were analyzed with Prism 6 (GraphPad Software, San Diego, Calif.).
Spinal -NPCs will be dissociated into a single-cell suspension at a concentration ranging from 1×10 {circumflex over ( )}4 to 1×10{circumflex over ( )}6 cells/μl in any vehicle (can be saline or any FDA approved vehicle) or cell can be encapsulated in a biomaterial/scaffold or matrix to fill in the lesion cavity. Cells will be stereotactically injected into spinal cord in defined rate
In revision, Submitted.
This is a patent cooperation treaty application which claims the benefit of 35 U.S.C. § 119 based on the priority of U.S. Provisional Patent Application No. 63/075,575 filed Sep. 8, 2020, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051239 | 9/8/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63075575 | Sep 2020 | US |
