Patent Application
20190151367
This document provides materials and methods for treating a damaged optic nerve in a mammal comprising administering a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, materials and methods for determining a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, and materials and methods for screening for factors that enhance maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell or cells.
Across the United States, approximately 400,000 people have multiple sclerosis (MS), and according to the National Multiple Sclerosis Society approximately 200 new cases are diagnosed every week. Many of these cases involve young people (18-45 years of age), especially females, struck at the peak of their economic and societal productivity. Optic neuritis is the heralding symptom in 15-20% of patients with MS and is associated with a 30-fold increase in the risk of developing MS. Nearly 80% of MS patients experience optic neuritis during the disease, with unilateral or bilateral attacks that result in permanent reduction or loss of vision in one or both eyes in 40-60% of patients. While some patients recover central vision, one third of affected eyes exhibit persistent visual impairment that includes reduced contrast sensitivity and problems with motion processing and depth perception. Almost half of patients with transient optic neuritis have a recurrent attack within 10 years. Median visual acuity of the affected eye in patients with optic neuritis is 20/60 and optic nerve atrophy can be measured within weeks following onset of the inflammatory attack. Moreover, the prevalence of persistent visual complaints in MS patients has been estimated at around 35%, and even MS patients without a clinical history of optic neuritis exhibit poor visual function, including diffuse visual field defects, reduced low-contrast acuity, impaired depth and motion perception, and decreased color-sensitivity. Pathophysiologically, restoration of vision in these patients requires preservation of the optic nerve axons and repair of the myelin sheath that confers such protection and facilitates high-speed, coordinated impulse conduction from the retina to higher visual processing centers within the brain. Currently, the therapies that exist to treat patients with demyelinating optic neuritis are purely palliative and almost exclusively immunomodulatory.
This document provides materials and methods for treating a damaged optic nerve in a mammal. For example, this document provides materials and methods for identifying a population of induced pluripotent stem cell-derived (iPSC-derived) oligodendrocyte precursor cells (OPCs) as having a remyelination potential quotient (RPQ) greater than about 25% (e.g., more than 1 in 4 cells acquire a phenotype of a mature myelinating oligodendrocyte) and administering the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, to the mammal. As described herein, a mammal having a damaged optic nerve can be effectively treated with a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a sufficiently high remyelination potential quotient.
In general, one aspect of this document features a method for treating a damaged optic nerve in a mammal. The method comprises, or consist essentially of, (a) identifying said mammal as having a condition of the optic nerve comprising optic nerve demyelination, (b) identifying a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells as having a remyelination potential quotient greater than about 25 percent, and (c) administering said population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to said mammal. The mammal can be a human. The population can be identified as having a remyelination potential quotient greater than about 25 percent by culturing a first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells in a microfluidic device comprising first and second microfluidic chambers, wherein the first microfluidic chamber comprises a neuron cell body of a cortical neuron, wherein the second microfluidic chamber comprises an axon of the cortical neuron, and wherein the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells is co-cultured with the axon in the second microfluidic chamber. The population can be identified as having a remyelination potential quotient greater than about 25 percent by determining the number of cells of the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of cells of the first portion by the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the second microfluidic chamber. The characteristic of a mature, myelinating oligodendrocyte can be selected from the group consisting of: a morphological characteristic, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, expression of a GST-pi mRNA, and combinations thereof. The remyelination potential quotient can be determined to be sufficient for administration of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to the mammal if the remyelination potential quotient is about 30 percent or higher.
The microfluidic device can further comprise a third microfluidic chamber, wherein the second microfluidic chamber comprises a segment of said axon, wherein the third microfluidic chamber comprises a distal end of the axon, and wherein a second portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells is co-cultured with the distal end of the axon in the third microfluidic chamber. The population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be identified as having a remyelination potential quotient greater than about 25 percent by determining the number of cells of the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of cells of the first portion by the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the second microfluidic chamber. Additionally or alternatively, the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined to have a remyelination potential quotient greater than about 25 percent by determining the number of cells of the second portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of cells of the second portion by the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the third microfluidic chamber. The characteristic of a mature, myelinating oligodendrocyte can be selected from the group consisting of: a morphological characteristic, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, expression of a GST-pi mRNA, and combinations thereof. The remyelination potential quotient can be determined to be sufficient for administration of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to the mammal if the remyelination potential quotient is about 30 percent or higher.
Administering can comprise intravitreal injection of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. The mammal can have a condition comprising multiple sclerosis, demyelinating optic neuritis, or both. Administering can drive remyelination of the optic nerve, restore axonal conduction, or both.
Another aspect of this document features a method for determining a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. The method comprises, or consist essentially of, culturing a first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells in a microfluidic device comprising first and second microfluidic chambers, wherein the first microfluidic chamber comprises a neuron cell body of a cortical neuron, wherein the second microfluidic chamber comprises an axon of the cortical neuron, and wherein the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells is co-cultured with the axon in the second microfluidic chamber. The remyelination potential quotient can be determined by determining the number of cells of the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of cells of the first portion by the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the second microfluidic chamber. The characteristic of a mature, myelinating oligodendrocyte can be selected from the group consisting of: a morphological characteristic, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, expression of a GST-pi mRNA, and combinations thereof. The remyelination potential quotient can be determined to be sufficient for administration of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to the mammal if the remyelination potential quotient is about 30 percent or higher.
The microfluidic device can further comprise a third microfluidic chamber, wherein the second microfluidic chamber comprises a segment of the axon, wherein the third microfluidic chamber comprises a distal end of the axon, and wherein a second portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells is co-cultured with the distal end of the axon in the third microfluidic chamber. The remyelination potential quotient can be determined by determining the number of cells of the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of cells of the first portion by the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the second microfluidic chamber. Additionally or alternatively, the remyelination potential quotient can be determined by determining the number of cells of the second portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of cells of the second portion by the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the third microfluidic chamber. The characteristic of a mature, myelinating oligodendrocyte can be selected from the group consisting of: a morphological characteristic, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, expression of a GST-pi mRNA, and combinations thereof. The remyelination potential quotient can be determined to be sufficient for administration of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to the mammal if the remyelination potential quotient is about 25 percent or above.
Another aspect of this document features a method for screening for factors that enhance maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell. The method comprises, or consist essentially of, culturing the induced pluripotent stem cell-derived oligodendrocyte precursor cell in a microfluidic device comprising first and second microfluidic chambers, wherein the first microfluidic chamber comprises a neuron cell body of a cortical neuron, wherein the second microfluidic chamber comprises an axon of the cortical neuron, wherein the induced pluripotent stem cell-derived oligodendrocyte precursor cell is co-cultured with the axon in the second microfluidic chamber, providing a first test factor to the second microfluidic chamber, and determining the maturation or myelination efficiency of the induced pluripotent stem cell-derived oligodendrocyte precursor cell in the second microfluidic chamber. The microfluidic device can further comprise a third microfluidic chamber, wherein the second microfluidic chamber comprises a segment of the axon, wherein the third microfluidic chamber comprises a distal end of the axon, and wherein a second induced pluripotent stem cell-derived oligodendrocyte precursor cell is co-cultured with the distal end of the axon in the third microfluidic chamber, providing a second test factor to the third microfluidic chamber, and determining the maturation or myelination efficiency of the induced pluripotent stem cell-derived oligodendrocyte precursor cell in the third microfluidic chamber. The maturation or myelination efficiency can be determined by determining a characteristic of a mature, myelinating oligodendrocyte selected from the group consisting of: a morphological characteristic, a functional characteristic, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, expression of a GST-pi mRNA, and combinations thereof. The maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell cultured in the presence of the factor can be increased compared to the maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell cultured in the absence of the factor.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
This document provides materials and methods for treating a damaged optic nerve in a mammal. For example, this document provides materials and methods for identifying a population of induced pluripotent stem cell-derived (iPSC-derived) oligodendrocyte precursor cells (OPCs) as having a remyelination potential quotient (RPQ) greater than about 25 percent.
As described herein, a mammal having a damaged optic nerve can be effectively treated with a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a sufficiently high RPQ (e.g., an RPQ greater than about 25, 35, 45, 50, 55, 65, or 75 percent). Any appropriate mammal having a damaged optic nerve can be treated as described herein. For example, humans and non-human primates such as monkeys can be identified as having a damaged optic nerve and treated with a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to drive remyelination of the optic nerve, restore axonal conduction, or both. In some cases, dogs, cats, horses, cows, pigs, sheep, mice, and rats can be identified and treated with a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells as described herein.
Any appropriate type of damage to the optic nerve can be treated in accordance with materials and methods provided herein. For example, damage to the optic nerve to be treated in accordance with materials and methods provided herein in the mammal can be caused by multiple sclerosis (MS). In some cases, the mammal can have a condition comprising multiple sclerosis, demyelinating optic neuritis, or both. In some cases, damage to the optic nerve to be treated in accordance with materials and methods provided herein can be caused by demyelination of the optic nerve induced by infiltrating inflammatory effector cells resulting in transient disruption of axonal conduction followed by chronic slowing, mistiming, and stochastic failure of the visual impulses that are transmitted from the retina to higher-order visual processing centers in order to confer vision. Such demyelinated axons become susceptible to injury and transection mediated by cellular immune effectors, toxic inflammatory mediators, and intra-axonal metabolic dysregulation, resulting in permanent and irrecoverable loss of information transmission through the visual pathway.
Any appropriate method can be used to identify a mammal having a damaged optic nerve. For example, imaging techniques, techniques to test and analyze vision, and visual evoked potentials (VEPs) can be used to identify mammals (e.g., humans) having a damaged optic nerve.
In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined prior to administering the population to a mammal. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by culturing the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, in a microfluidic device comprising a plurality of microfluidic chambers, e.g. a first and second microfluidic chamber. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured in a microfluidic device with one or more neurons, e.g., one or more cortical neurons. In some cases, a single cortical neuron is cultured in a plurality of chambers of the microfluidic device. For example, a cortical neuron can be cultured in a microfluidic device such that the body of the cortical axon is cultured in one microfluidic chamber and the axon of the cortical neuron is cultured in one or more other microfluidic chambers that can be fluidically separated. In some cases, a cortical neuron can be cultured in a microfluidic device such that the body of the cortical axon is cultured in one microfluidic chamber, an axon is cultured in a second microfluidic chamber, and the distal end of the axon is cultured in third microfluidic chamber. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured with an axon in the same microfluidic chamber. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured en passant of an axon in the same microfluidic chamber. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured with the distal end of an axon in the same microfluidic chamber. By exploiting the microfluidic separation of the cell body chamber from the axon chamber or chambers, it is possible to introduce different media formulations into the separate chambers.
In some cases, a remyelination potential quotient of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the number of cells of a first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells having a characteristic of a mature, myelinating oligodendrocyte and dividing the number of mature cells of the first portion by the total number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the same microfluidic chamber. For example, if 80% of the cells of the first portion of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells have a characteristic of a mature, myelinating oligodendrocyte, the RPQ of the population is 80%. A remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be similarly determined in other embodiments described herein.
In some cases, a microfluidic device used to assess cells capable of effectively treating a damaged optic nerve in a mammal can have three microfluidic chambers. In some cases, a neuron, e.g., a cortical neuron, can be cultured in such a three-chambered microfluidic device. For example, the body of a neuron can be cultured in a first chamber, an axon of the neuron can be cultured in a second chamber, and the distal end of the axon of the neuron can be cultured in a third chamber. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured with: 1) a segment of an axon of the neuron being cultured in the second chamber, 2) the distal end of the axon of the neuron being cultured in the third chamber, or 3) both. In some cases, the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the number of cells of the portion of the population co-cultured en passant of the axon being cultured having a characteristic of a mature, myelinating oligodendrocyte and dividing that number by the total number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the second microfluidic chamber. In some cases, the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the number of cells of the portion of the population co-cultured with a distal end of an axon of the neuron being cultured having a characteristic of a mature, myelinating oligodendrocyte and dividing that number by the total number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the third microfluidic chamber.
Any suitable characteristic or characteristics of mature, myelinating oligodendrocytes can be used to determine the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. Examples of such characteristics include, without limitation, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, and expression of a GST-pi mRNA. In some cases, a characteristic of a mature, myelinating oligodendrocyte can be a morphological characteristic. For example, a morphological characteristic of a mature, myelinating oligodendrocyte can be the making of one or more contacts with one or more axons of a cortical neuron, and branched morphology with axon ensheathment. In some cases, a characteristic of a mature, myelinating oligodendrocyte can be a functional characteristic. For example, a functional characteristic of a mature, myelinating oligodendrocyte can be myelinating or remyelinating activity on an axon of a cortical neuron. In some cases, the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the number of cells of a population, or portion thereof, having one or more characteristics selected from a plurality of characteristics of a mature, myelinating oligodendrocyte, e.g. an expression characteristic or characteristics, a morphological characteristic or characteristics, and/or a functional characteristic or characteristics of a mature, myelinating oligodendrocyte.
In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined to be sufficient for administration of the population to the mammal if the remyelination potential quotient is above a certain threshold. For example, suitable remyelination potential quotient threshold for a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be greater than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%70%, 75%, 80%, 85%, 90%, or 95%. In general, the higher the RPQ of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, the more suitable that population is for administration to a mammal.
In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells for use in treating a damaged optic nerve of a mammal (e.g., a human) can be determined by determining the remyelination potential quotient of two or more subpopulations of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells according to one or more methods described herein. For example, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the RPQ of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more subpopulations. In some cases, a RPQ of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the RPQ of two or more subpopulations using two or more microfluidic devices as described herein, e.g., two or more microfluidic devices having three microfluidic chambers, wherein a neuron, e.g., a cortical neuron, is co-cultured in the two or more microfluidic devices with the two or more subpopulations of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by co-culturing two or more subpopulations of the population in two or more microfluidic devices, wherein each of the subpopulations is co-cultured en passant of an axon, e.g., an axon of a cortical neuron. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by co-culturing two or more subpopulations of the population in two or more microfluidic devices, wherein each of the subpopulations is co-cultured with a distal end of a neuron, e.g., a cortical neuron. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by averaging the determined remyelination potential quotients of two or more subpopulations of the population. In some cases, a coefficient of variance for log-normalized remyelination potential quotient values of each subpopulation can be calculated as a measure of reproducibility.
In some cases, RPQ of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be clonally derived from a single pluripotent stem cell-derived oligodendrocyte precursor cell. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be derived from heterogeneous starting population of a pluripotent stem cell-derived oligodendrocyte precursor cells.
In some cases, once a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells is determined to have a suitable remyelination potential quotient, aliquots of that population can be frozen and stored for future use.
Any suitable route of administration can be used in accordance with methods of treating a damaged optic nerve in a mammal, as provided herein. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered to the mammal via an intravitreal injection, e.g. injection into the vitreous proximal to the optic nerve head. A population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to be injected into a mammal can be formulated with any number of acceptable carriers, fillers, and/or vehicles.
Effective numbers of induced pluripotent stem cell-derived oligodendrocyte precursor cells in an administered population can vary depending on the severity of the damage to the optic nerve, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. In some cases, from about 1×107 to about 5×107 cells/eye can be administered to a mammal (e.g., a human). In some cases, from about 107 to about 108, from about 106 to about 107, from about 105 to about 106, from about 104 to about 105, from about 103 to about 104 cells/eye can be administered to a mammal (e.g., a human).
If a particular mammal fails to respond to a particular number of induced pluripotent stem cell-derived oligodendrocyte precursor cells, the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be increased. In some cases, the number of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be increased by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more. After receiving such an increased number of induced pluripotent stem cell-derived oligodendrocyte precursor cells, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. The effective number of induced pluripotent stem cell-derived oligodendrocyte precursor cells can remain constant or can be adjusted as a sliding scale or variable numbers depending on the mammal's response to treatment. Various factors can influence the actual effective number of induced pluripotent stem cell-derived oligodendrocyte precursor cells used. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., damage to an optic nerve) may require an increase or decrease in the actual effective number of induced pluripotent stem cell-derived oligodendrocyte precursor cells administered.
In some cases, for humans, a population of autologous induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered. For non-human mammals, a population of autologous or homologous induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered.
In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered once to a mammal. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered more than once to a mammal.
In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered to a mammal (e.g., a human) in combination with one or more additional therapeutic agents. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered to a mammal in combination with valproic acid. In some cases, a combination therapy of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells and valproic acid can enhance OPC recruitment, survival, and/or cumulative myelination compared to either therapy alone. In some cases, such one or more additional therapeutic agents can be blood-brain barrier permeable drugs. Examples of suitable blood-brain barrier permeable drugs include, without limitation, miconazole, clobetasol, and benztropine. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered to a mammal simultaneously with one or more additional therapeutic agents. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered to a mammal prior to administration of one or more additional therapeutic agents. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be administered to a mammal after administration of one or more additional therapeutic agents.
In some cases, a course of treatment, the damage to or function of an optic nerve present within a mammal, and/or the severity of one or more symptoms related to the condition being treated (e.g., damage to an optic nerve) can be monitored. Any appropriate method can be used to determine whether or not damage to an optic nerve of a mammal is reduced and/or whether the function of the optic nerve is improved. For example, imaging techniques, techniques to test and analyze vision, and visual evoked potentials (VEPs) can be used to determine whether or not damage to an optic nerve of a mammal (e.g., a human) is reduced and/or whether the function of the optic nerve of a mammal (e.g., a human) is improved. In some cases, one or more of evoked potential amplitude, latency, peak pulse-width, and/or number of missed responses can be measured and used to determine whether or not damage to an optic nerve of a mammal is reduced and/or whether the function of the optic nerve is improved. In some cases, treating a mammal (e.g., a human) in accordance with a method provided herein can drive remyelination of the optic nerve, restore axonal health, or both.
This document also provides materials and methods for determining a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. For example, this document provides materials and methods for culturing the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or a portion thereof, in a microfluidic device comprising a plurality of microfluidic chambers. As described herein, the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured in one or more of the plurality of microfluidic chambers with an axon of a neuron, e.g., a cortical neuron, and the remyelination potential quotient of the population or portion can be determined.
Any suitable device (e.g., a microfluidic device) or method described herein of determining a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells for treating a damaged optic nerve in a mammal can be used to determine a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells generally. For example, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured with a neuron, e.g., a cortical neuron, in a microfluidic device comprising three chambers. For example, the body of a neuron can be cultured in a first chamber, an axon of the neuron can be cultured in a second chamber, and the distal end of the axon of the neuron can be cultured in a third chamber. In some cases, a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or portion thereof, can be co-cultured with: 1) an axon of the neuron being cultured in the second chamber, 2) the distal end of the axon of the neuron being cultured in the third chamber, or 3) both. In some cases, the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the number of cells of the portion of the population co-cultured en passant of an axon being cultured having a characteristic of a mature, myelinating oligodendrocyte and dividing that number by the total number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the second microfluidic chamber. In some cases, the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the number of cells of the portion of the population co-cultured with a distal end of an axon of the neuron being cultured having a characteristic of a mature, myelinating oligodendrocyte and dividing that number by the total number of induced pluripotent stem cell-derived oligodendrocyte precursor cells introduced into the third microfluidic chamber.
In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the remyelination potential quotient of two or more subpopulations of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells according to more or more methods described herein. For example, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the remyelination potential quotient of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more subpopulations. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by determining the remyelination potential quotient of two or more subpopulations using two or more microfluidic devices as described herein, e.g., two or more microfluidic devices having three microfluidic chambers, wherein a neuron, e.g., a cortical neuron, is co-cultured in the microfluidic device with the two or more subpopulations of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by co-culturing two or more subpopulations of the population in two or more microfluidic devices, wherein each of the subpopulations is co-cultured en passant of an axon. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by co-culturing two or more subpopulations of the population in two or more microfluidic devices, wherein each of the subpopulations is co-cultured with a distal end of a neuron, e.g., a cortical neuron. In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by averaging the determined remyelination potential quotient of two or more subpopulations of the population. In some cases, a coefficient of variance for log-normalized remyelination potential quotient values of each subpopulation can be calculated as a measure of reproducibility.
In some cases, a remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined to be sufficient for administration of the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells to the mammal if the remyelination potential quotient is above a certain threshold. For example, suitable remyelination potential quotient threshold for a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be greater than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%70%, 75%, 80%, 85%, 90%, or 95%. In general, the higher the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, the more suitable that population is for administration to a mammal.
This document also provides materials and methods for screening for factors that enhance maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell or a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. For example, this document provides materials and methods for culturing the population of induced pluripotent stem cell-derived oligodendrocyte precursor cells, or a portion thereof, in a microfluidic device comprising a plurality of microfluidic chambers. As described herein, an induced pluripotent stem cell-derived oligodendrocyte precursor cell or cells can be co-cultured in one of the plurality of microfluidic chambers with an axon (e.g., en passant of the axon, distal end of the axon, or both of a neuron, e.g., a cortical neuron, a test factor can be provided to that chamber, and the effect of the test compound on maturation or myelination efficiency of the induced pluripotent stem cell-derived oligodendrocyte precursor cell or cells can be determined.
Any microfluidic device or method of using such a device described herein can be used to screen for factors that enhance maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell or a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells.
In some cases, a single factor can be screened for its effect and/or its enhancement of maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell or a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells. In some cases, a plurality of factors can be screened for their effect and/or their enhancement of maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell or a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells.
Maturation or myelination efficiency can be determined by assaying for any of a variety of characteristics of a mature, myelinating oligodendrocyte. Examples of such characteristics include, without limitation, expression of a MOG polypeptide, expression of a CC1 polypeptide, expression of a MBP polypeptide, expression of a PLP polypeptide, expression of a MAG polypeptide, expression of a GST-pi polypeptide, expression of a MOG mRNA, expression of a CC1 mRNA, expression of a MBP mRNA, expression of a PLP mRNA, expression of a MAG mRNA, and expression of a GST-pi mRNA. In some cases, a characteristic of a mature, myelinating oligodendrocyte can be a morphological characteristic. For example, a morphological characteristic of a mature, myelinating oligodendrocyte can be the making of one or more contacts with one or more axons of a cortical neuron, and branched morphology with axon ensheathment. In some cases, a characteristic of a mature, myelinating oligodendrocyte can be a functional characteristic. For example, a functional characteristic of a mature, myelinating oligodendrocyte can be myelinating or remyelinating activity on an axon of a cortical neuron. In some cases, a factor (or factors) can be screened for its effect and/or its enhancement of maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell or a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells by determining whether cells or cells have one or more characteristics selected from a plurality of characteristics of a mature, myelinating oligodendrocyte, e.g. an expression characteristic or characteristics, a morphological characteristic or characteristics, and/or a functional characteristic or characteristics of a mature, myelinating oligodendrocyte.
In some cases, maturation or myelination efficiency can be determined by culturing a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells with one or more factors, and administering the population, or portion thereof, to an animal (e.g., a mouse, a rat, a primate, a non-human mammal, a dog, a cat, a horse, a cow, a pig, or a sheep) and harvesting and analyzing an optic nerve from the animal. In some cases, an optic nerve is harvested at 1, 2, 3, 4, 5, 6, 7, 14, and/or 21 days post-transplant. In some cases, static analysis of remyelination of harvested optic nerves is accomplished using histology, confocal microscopy, and/or cross-sectional electron microscopy.
Robust survival and myelination of chambered axons by induced pluripotent stem cell-derived oligodendrocyte precursor cells can require a compromise between the nutrient and support factor needs of the axons, the oligodendrocyte precursor cells, and the maturing oligodendrocytes. In some cases, media conditions necessary to maintain healthy axons and fluid dynamics involved in media replenishment and ongoing provision of growth factors can be optimized. In some cases, fluidic shear stress during the addition of factors or cells to microfluidic chambers can be minimized. In some cases, by exploiting the microfluidic separation of the cell body chamber from one or more axon chambers, different media formulations can be introduced into the separate chambers and oligodendrocyte precursor cells survival and differentiation are increased. In some cases, timing of OPC introduction into the axon chambers is optimized and standardized. In some cases, frequency of media replenishment and the chemical components of the axon chamber media formulation can be optimized and standardized. In some cases, chamber media for OPC differentiation can include: DMEM/F12 w/o HEPES or phenol red; 1.25×B-27 Supplement, serum free; 0.25×N1 Medium Supplement; 0.5×N-2 Supplement; 30 ng/mL T3; 27.5 μM 2-mercaptoethanol; 2.5 ng/mL NT3; 50 ng/mL biotin; 0.5 μM dibutyryl cAMP; 2.5 ng/mL PDGF-AA; 2.5 ng/mL IGF-1; and 5 ng/mL BDNF. In some cases, methods and materials provided herein can be used to test factors, e.g., mitogens, signaling molecules, and oligo-inductive factors, for their effect on OPC differentiation. Examples of such factors include, without limitation, those listed in Table 1.
In some cases, tested and optimized culture conditions can be assessed for efficacy as compared to mouse OPCs derived by shake-off from mixed glia cultures (i.e. non-iPSC-derived). In some cases, myelination is assessed by: 1) confocal and super-resolution microscopy and quantification of 3D reconstructed GFP-positive membrane structures, 2) transmission EM and analysis of myelin wrapping morphology, including g ratio, 3) RTPCR analysis of myelin gene expression levels in the axon chamber, and/or 4) Western blot analysis of myelin protein expression levels in the axon chamber. In some cases, optimized and standardized media conditions can increase the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells.
In some cases, quantitative robustness of the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be assessed by altering the density of iPSC-derived OPCs added to the axon chamber in order to systematically change the ratio of OPCs to axons (ROTA). In some cases, the remyelination potential quotient of a population of induced pluripotent stem cell-derived oligodendrocyte precursor cells can be determined by measuring the RPQ when the ROTA is sufficient to maximize, but not exceed, the axonal space available for myelination.
In some cases, maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell cultured in the presence of a factor or factors can be increased compared to maturation or myelination efficiency of an induced pluripotent stem cell-derived oligodendrocyte precursor cell cultured in the absence of a factor or factors.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
MFCs were prepared as described elsewhere (Sauer et al., Neurobiol. Dis., 59:194-205 (2013)). Briefly, silicone elastomer (Sylgard 184) base and curing agent (mixed 10:1) were poured over etched fused silica molds. MFCs were placed in a vacuum chamber and incubated at 37° C. before being cut out from the molds and sterilized for use. In a sterile hood, acid washed and sterile coverslips (22×22 mm) were placed in 6-well tissue culture plates and coated with 0.5 mg/mL poly-ornithine overnight at 37° C. Prior to plating cells, the printed surface of the sterilized MFCs was adhered to glass cover slips to achieve a leak-proof chamber.
Primary murine cortical neuron cultures were prepared as described elsewhere (Sauer et al., Neurobiol. Dis., 59:194-205 (2013)). Briefly, cells were obtained from embryonic day 15 B6 mouse cortices and plated at 1.5×105 cells per microfluidic chamber with half media changes on alternate days. After verification of axonal growth through the microgrooves (day in vitro (DIV) 4-5)), aliquots of OPCs to be tested were plated into the middle chamber of the MFC and co-cultured for up to two weeks with differentiation factors.
Following fixation in the MFC with 4% paraformaldehyde, cells were stained to determine the extent of myelination. Cells were incubated with blocking buffer (5% serum of secondary-antibody host species, 1% BSA, 0.1% Triton-X in DPBS containing Ca2+ and Mg2+) for 60 minutes at room temperature followed by overnight incubation with primary antibodies at 4° C. (PLP: Millipore, MAB388, 1:200; MOG: Millipore, MAB5680 1:100; MBP: Millipore, MAB386, 1:150; APC/CC1: Abcam, ab15270, 1:250; NF: Sternberger, SMI-312R, 1:750). After extensive washing, cells were stained with appropriate secondary antibodies, counterstained with DAPI, and mounted onto glass slides.
Cuprizone is a copper chelator, which causes apoptosis of mature oligodendroglia, followed by microglial recruitment, and phagocytosis of myelin. Demyelination was induced by feeding mice a diet containing 0.3% cuprizone for 9 weeks. When fed on this diet, mice exhibit demyelination in a well-characterized series of events. Peak demyelination occurred at 6-7 weeks with spontaneous remyelination occurring 2-4 weeks after transition to regular diet. Efficacy of OPC transplants upon reintroduction of regular diet was evaluated. Assessment of remyelination following transplant was performed as described elsewhere (Deb et al., PLoS One, 5:e12478 (2010)). Briefly, semi-thin sections of optic nerves were stained with para-phenylenediamine, imaged at 100× magnification following a predetermined sampling scheme, and analyzed using automated ImageJ macros to measure myelinated fibers.
Maintaining a sterile field, 1.0 μL of media containing >104 OPCs were injected into the vitreous humor of anesthetized mice, using a custom 34-gauge needle mounted on a 5 μL Hamilton syringe. Despite the small dimensions of the murine eye and the high needle gauge, OPCs were consistently and reproducibly delivered into the vitreous cavity with this method, and transferred cells were imaged using fluorescent fundus imaging of the mouse eye (evidence from >30 mice; data not included) (
Image-guided optical coherence tomography (OCT): OCT was an imaging technique based on light interferometry which can be used to capture images from biomedical tissue at micrometer resolution. An image guided OCT system (Micron III, Phoenix Labs, USA) with a resolution of 4 μm, similar to human OCT scanners available today, was used. The system had the capability to capture bright-field as well as 3 channel fluorescent retinal images (
To measure the functional outcomes of OPC transplantation, VEPs were recorded. Intracranial supradural screw electrodes (plastics1.com 8L0X3905201F) were implanted over the visual cortex as described elsewhere (Deb et al., (2010) PLoS One 5:e12478) (
For collection of optic nerves and brain tissue, mice were perfused with 4% paraformaldehyde or Trump's fixative, and CNS tissues were post-fixed in the respective solution for 24 hours.
Following adequate fixation, whole optic nerves and retinas were transferred to a 48 well dish and blocked for 2 hours in PBS containing 5% serum of secondary-antibody host species, 1% BSA, and 0.1% Triton-X. For tissues intended for clearing and whole tissue imaging, the amount of Triton-X was raised to 1%. After blocking, optic nerves were incubated overnight at 4° C. with primary antibodies (MOG: Millipore, MAB5680 1:100; MBP: Millipore, MAB386, 1:150; APC: Abcam, ab15270, 1:250). Tissues were washed extensively, stained for 2 hours with appropriate secondary antibodies, counterstained with DAPI, and mounted onto glass slides for microscopy or moved to clearing reagents.
Optic nerves were cleared using a modification of a ScaleSQ protocol as described elsewhere (Hama H et al., (2015) Nat Neurosci 18:1518-1529). Briefly, tissues were placed in dextro-sorbitol (25% w/v), urea (9.5M), and Triton-X 100 (3% w/v) in distilled water at 37° C. overnight and subsequently in dextro-sorbitol (40% w/v), urea (4M), glycerol (15% w/v), and DMSO (20% v/v) in distilled water for 5 hours. After visual verification of tissue clarity, optic nerves were mounted on glass cover slides for image acquisition.
Fluorescence and bright field images of cultured cells and slide-mounted whole retina and optic nerve tissues were imaged with a LSM 780 inverted confocal microscope, an upright two photon microscope (Olympus FV1000MPE), or an inverted Axio Observer Z1 microscope equipped with Apotome.
Murine cortical neurons were successful cultured in microfluidic chambers in order to isolate the neuronal cell bodies from their axons. Microgrooves within a microfluidic device prevented cell bodies from entering the axonal chamber, thereby allowing easier visualization and manipulation of axons without the interference of neuronal cell bodies and other cells types (such as oligodendrocytes and astrocytes) that are present at plating (
Cell populations enriched for OPCs were created from murine lines as described elsewhere (Terzic et al., Cell Transplantation, 25(2):411-424 (2016)).
Utilizing a three-chamber MFC system, mouse iPSC-derived OPCs were successfully co-cultured with primary cortical neuron axons. The OPCs became GFP+ oligodendrocytes and a subset made extensive branching contacts with neuronal axons (
In a separate experiment utilizing a three-chamber MFC system, OPCs were co-cultured with neuronal axons for up to 14 days, and serial longitudinal imaging of OPCs was performed. By 11 days in culture, OPCs exhibited the phenotype of a mature oligodendrocyte, making multiple contacts with multiple axons (
These results demonstrated successfully co-culturing iPSC-derived OPCs in a MFC three-chamber platform, and also demonstrated the ability to quantify the myelinating capacity of OPCs prior to transplantation.
Cuprizone is a copper chelator that causes apoptosis of mature oligodendroglia, followed by microglial recruitment and phagocytosis of myelin. Demyelination was induced by feeding mice a diet containing 0.3% cuprizone for 9 weeks. When fed on this diet, mice exhibit demyelination in a well-characterized series of events. Peak demyelination occurs at 6-7 weeks with spontaneous remyelination occurring 2-4 weeks after transition to regular diet. PPD staining of thin sections from araldite-embedded optic nerve revealed the pattern of normal myelination in the optic nerve of animals fed control chow (
Semi-thin sections from araldite-embedded optic nerves were stained with PPD (p-phenylenediamine). The staining revealed the pattern of normal myelination in the optic nerve of animals fed control chow (
To further validate the in vitro findings above, GFP+ OPCs were transplanted into the vitreous cavity of mice (
Mice were subjected to electrode implantation surgery and either received OPC transplant or served as controls (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Application Ser. No. 62/317,839, filed on Apr. 4, 2016. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/025701 | 4/3/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62317839 | Apr 2016 | US |
