Expansion and differentiation of neuronal precursor cells

Abstract

The present invention relates to the production of neuronal progenitor cells, a composition containing the same, and therapeutic uses.

Description

Technology Field

[0001] The present invention relates to the preparation of neural progenitor cells, compositions comprising the same, and therapeutic uses thereof. Background Technology

[0002] Reference to prior art indicated in the specification does not constitute an acknowledgment or suggestion that the prior art forms part of ordinary knowledge in any area, or that a person skilled in the art would reasonably expect to understand that the prior art is correlated with and / or can be combined with other prior art.

[0003] Multifunctional stem cells create new nerve cells ( de novo This is the conventional starting point for manufacturing—leading to the expansion of cells in their primitive replicative state, followed by differentiation into a mature neurophenotype. Multifunctional stem cells can be isolated from embryonic-derived cells (i.e., embryonic stem cells) or adult stem cell repositories (i.e., adult stem cells such as mesenchymal stem cells), or, as discovered more recently, by reprogramming mature post-differentiated cells (e.g., fibroblasts) into embryonic-like stem cells (i.e., induced pluripotential stem cells).

[0004] By definition, each of these methods has a general limitation in that it can result in various cell fates (i.e., multifunctional). Therefore, the yield of neurons is low and variable, and results in non-neuronal phenotypes even after treatment under specific neuronal differentiation conditions. For example, glial cell differentiation is a common limitation after such treatment. For biological research, commercial applications, or therapeutic uses, it would be useful to be able to produce a uniform population of monodifferentiated neural progenitor cells—that is, a constant, transiently replicable, and neuronal lineage-determined population.

[0005] The closest approach today is the direct genetic reprogramming of mature differentiated cells (e.g., fibroblasts) into post-mitotic neurons without going through a proliferative stem or progenitor cell stage via the upregulation of major neuronal induction genes (Vierbuchen et al., 2010; Pang et al., 2011; Son et al., 2011; Zhou et al., 2014; Tsunemoto et al., 2018). However, this method relies on genetic manipulation and does not produce scalable populations, and, like all other methods, implies the problem of unacceptable high cell-to-cell-line variability (Truong et al., 2016).

[0006] In light of this, it is interesting that human skin and the central nervous system share the same embryonic origin, which is an ectoderm germline. Adult stem cells and progenitor cells in these two organs share many of the same cell markers (e.g., Nestin nestin), CD133, Sox2 , etc.)) express and many such cell cycle regulators (( Nogin ( Noggin), SHH, FGF, EGF, BDNF They are hardly aware that they use , etc.

[0007] In 2001, Toma et al. (2001) first demonstrated that pluripotent stem cells in mammalian hair follicle niches could produce neurons in vitro, even though the yield after differentiation was low (15%) and most cells evolved into non-neuronal types. This fundamental result was subsequently replicated using human skin by this group (Vierbuchen et al., 2010; Pang et al., 2011; Son et al., 2011; Zhou et al., 2014; Tsunemoto et al., 2018) and other groups (Joannides et al., 2004; Yu et al., 2010; Yu et al., 2006; Belicchi et al., 2004). Interestingly, the reverse has also been demonstrated recently: Hwang et al. (2016) discovered that neural stem cell extracts (of human fetal origin) can actually increase hair regrowth in vivo in depilated mice.

[0008] Therefore, while these two organ systems differ extensively in structure and function, they share many of the biological mechanisms governing stem cell and progenitor cell proliferation. Furthermore, stem cells and progenitor cells in one niche can infer the role and identity of the other based solely on environmental signals. Given the difficulty of accessing adult human neural stem cells from the physiological niche (the brain), it is conceptually attractive that neural progenitor cells can be derived from the individual's own hair follicle progenitor cell population without reclassification for genetic manipulation within that individual.

[0009] So far, this premise has actually failed. Cell viability and the yield of nerve cells from natural human skin were too low, and the variability between cell lines was too large.

[0010] The progenitor cell source in this context—adult human hair follicles—contains both monofunctional progenitor cells and pluripotent stem cells. Monofunctional progenitor cells differ fundamentally from pluripotent stem cells in that they are destined to be limited to only a single cell lineage and cannot undergo unlimited replication in vitro. Recently, it has been revealed that there is significant heterogeneity among progenitor cells transiently amplified in hair follicles, and this variation enables the development of subpopulations with different lineage destinies (Yang et al., 2017). Furthermore, this "lineage infidelity" occurs maximally under wound repair, oncogenic transformation, or in vitro cell culture conditions (Ge et al., 2017; Fuchs, 2018).

[0011] What has not been recognized until now is that subpopulations or progenitor cells of human hair follicles possess the ability to contain limitations on potential neuronal fate.

[0012] The ability to isolate and expand such single-function neuronal precursors from mature skin varies widely among species. For example, we previously developed this process in the skin of adult cats (Valenzuela et al. (2008), but this method cannot generally be applied to adult skin because the quantity and quality of hair follicles vary significantly between individuals and within individuals. means of solving the problem

[0014] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0015] - Treat a hair follicle cell sample in a condition where the hair follicle cells can transition to the growth phase, thereby forming a conditioned cell sample;

[0016] - Treat a cell sample conditioned in a condition where the number of cells containing neuronal lineage biomarkers in the sample can be enhanced;

[0017] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0018] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0019] - Treat a hair follicle cell sample in a condition where the hair follicle cells can transition to the growth phase, thereby forming a conditioned cell sample;

[0020] - By treating a conditioned cell sample in a condition where neuroglobules can be formed from the cells of the conditioned cell sample;

[0021] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0022] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0023] - Treat a hair follicle cell sample in a condition where the hair follicle cells can transition to the growth phase, thereby forming a conditioned cell sample;

[0024] - By treating this conditioned cell sample in a condition where neuroglobules can be formed from the cells of the conditioned cell sample;

[0025] - A sample of neuroglobules provides conditions that expand the number of neuroglobules;

[0026] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0027] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0028] - Treat a hair follicle cell sample in a condition where the hair follicle cells can transition to the growth phase, thereby forming a conditioned cell sample;

[0029] - Provides a condition in which cells in a conditioned cell sample are separated into single cells;

[0030] - By treating this conditioned cell sample in a condition where neuroglobules can be formed from the cells of the conditioned cell sample;

[0031] - A sample of neuroglobules provides conditions that expand the number of neuroglobules;

[0032] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0033] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0034] - A skin sample, skin containing hair follicle cells, is treated in a condition in which hair follicle cells in the skin can transition to a growth phase, thereby forming a conditioned skin sample;

[0035] - Treat skin samples conditioned in a condition where the number of cells containing neuronal lineage biomarkers can be enhanced in the sample;

[0036] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0037] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0038] - A skin sample, skin containing hair follicle cells, is treated in a condition in which hair follicle cells in the skin can transition to a growth phase, thereby forming a conditioned skin sample;

[0039] - Depleting finally differentiated cells, apoptotic cells, or cell fragments from a conditioned skin sample to thereby form a non-terminally differentiated cell sample;

[0040] - Treat a sample of non-finally differentiated cells under conditions where the number of cells containing neuronal lineage biomarkers can be enhanced in the sample;

[0041] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0042] In one embodiment, a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing a neuronal lineage biomarker is provided, comprising:

[0043] - A skin sample, skin containing hair follicle cells, is treated in a condition in which hair follicle cells in the skin can transition to a growth phase, thereby forming a conditioned skin sample;

[0044] - Release cells from the conditioned skin as a sample;

[0045] - Depleting finally differentiated cells, apoptotic cells, or cell fragments from a conditioned skin sample to thereby form a non-terminally differentiated cell sample;

[0046] - Treat a sample of non-finally differentiated cells under conditions where the number of cells containing neuronal lineage biomarkers can be enhanced in the sample;

[0047] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0048] In a further embodiment, a composition of cells produced by the method described above is provided. This composition may comprise a therapeutically effective amount of cells and a pharmaceutically acceptable diluent, carrier, or excipient. The cellular component of this composition may consist of cells produced by the method described above, or the cellular component may comprise cells produced by the method described above and further comprise other cells. This composition may be modified to be injectable or infused.

[0049] In further embodiments, this cell composition is used for treatment, for example, for the treatment of conditions or diseases of nerve tissue. Therefore, in one embodiment, a method for treating a disease or condition, preferably a disease or condition of nerve tissue, is provided, comprising administering the composition described above to an individual requiring said treatment, thereby treating said disease or condition of said individual.

[0050] In a further embodiment, the cell composition described above is used to treat a disease or condition, preferably a disease or condition of nerve tissue.

[0051] In a further embodiment, the cell composition described above is used to manufacture a medicine for treating a disease or condition, preferably a disease or condition of nerve tissue.

[0052] In a further embodiment, one or more devices comprising the cell composition described above are provided for treating a disease or condition, preferably a disease or condition of nerve tissue.

[0053] As used herein, except where the context otherwise requires, the term “comprise” and variations of this term such as “comprising,” “comprises,” and “comprised” are not intended to further exclude additives, components, integers, or steps.

[0054] Further aspects of the present invention and further embodiments of the aspects described in the prior art will become apparent from the following description, which is given as an example of an embodiment. Brief explanation of the drawing

[0056] Fig. 1. Typical cell yield per hair follicle after isolating neural bulbs with or without chemical pretreatment with sonic hedgehog (SHH) for 6 or 72 hours. Data are from 45-year-old men. Fig. 2. Excellent cell yield after immediate enzymatic treatment and crushing of hair follicles with collagenase / dispase. Fig. 3.Brightfield micrographs of typical neuroglobules derived from hair follicles of a 45-year-old male (upper row) and a 25-year-old male (lower row). Fig. 4. Fluorescence micrographs of neurobulbs stained for a series of typical neural progenitor cell biomarkers. A) Very common neural stem cell marker Nestin (>90% of cells positive). B) Neural progenitor cell marker Doublecortin (DCX; >80% of cells positive). C) Immature neuron marker βIII-tubulin (B3T; >50% of cells positive). D) Negative control (no primary). All neurobulbs were derived from hair follicles of 34-year-old males. Fig. 5. Monolayer expansion of HFNs. Bright-field micrograph of the typical shape of fused HFNs obtained from a 25-year-old male. The fluorescence image shows the expression of the common (>90% positive) stem cell marker CD133 protein and the immature neuronal marker βIII-tubulin (βIII-TUBULIN) (B3T). The fluorescence image of HFN cells is from a 45-year-old male. Specific details for implementing the invention

[0057] The present invention provides a method for producing a neuronal progenitor cell composition that exhibits limited cell lines and variations between cell lines and has the ability to rapidly expand to a commercially useful number of cells. The method includes:

[0058] - Treat a hair follicle cell sample in a condition where the hair follicle cells can transition to the growth phase, thereby forming a conditioned cell sample;

[0059] - Treat a cell sample conditioned in a condition where the number of cells containing neuronal lineage biomarkers in the sample can be enhanced;

[0060] This includes a method for producing a composition of neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers.

[0061] The cells of the composition produced by this method are neural progenitor cells or, more generally, proliferating cells expressing neuronal lineage biomarkers. Neuronal lineage biomarkers cover a developmental spectrum that may include (most primarily) the neural stem cell-like marker Nestin, the radial glial cell marker GFAP, the neuroblastic markers doublecortin (DCX) and NCAM, and the immature neuron marker betaIII-tubulin. More common stem cell-like markers such as CD133, Sox2, and OCT4 may also be expressed. Cells positive for these markers are reinforced in the neuroglobules and have the potential to eventually form differentiated neurons. Generally, these cells are multifunctional, meaning they have the ability to produce the final differentiated cells of the nervous system, such as neurons and glial cells.

[0062] Generally, the initial step of this method involves the treatment of hair follicle cells. As described below, these cells can be obtained in the form of skin samples containing hair follicles. On the other hand, these cells can be obtained from hair follicle isolates. As is generally known, hair follicle cells can exist within phase ranges (anagen, catagen, and telogen) in the context of growth. Telogen is the resting phase (i.e., without growth), anagen is the growth phase, and catagen is an intermediate phase between anagen and telogen. The treatment step results in the enhancement of hair follicle cells in the growth phase or anagen phase. This enhancement can occur by stimulating hair follicle cells to cause cells in the telogen phase to transition to the anagen phase, and / or by preventing cells in the growth phase from transitioning to the catagen phase. As a result of the treatment step, hair follicle cells possessing the potential to develop neuronal lineage biomarkers in the growth phase are specifically enhanced.

[0063] In a further step, cells are treated to enhance, or otherwise increase, the number of cells containing neuronal lineage biomarkers. This can be achieved by treating to increase the number of cells containing neuronal lineage biomarkers and / or by decreasing the number of cells not containing neuronal lineage biomarkers. In a particularly preferred step, cells are treated under conditions that enable the formation of neuroglobules. Methods for forming neuroglobule cells are well known in the art and are further illustrated herein by example. In one embodiment, cells are treated to enhance, or otherwise increase, the number of cells containing neuronal lineage biomarkers, or are treated to enable neuroglobule formation by culturing the cells in a bioreactor. A bioreactor may be used to provide enhanced conditions for neuroglobule formation.

[0064] A first embodiment of the present invention is described herein, wherein a skin sample containing hair follicles is used to induce a composition of neuronal progenitor cells, wherein the skin sample containing hair follicles is treated under conditions that allow hair follicle cells in the skin to transition to a growth phase, thereby forming conditioned skin; and thereafter, the conditioned cell sample is treated under conditions that allow the number of cells containing neuronal lineage biomarkers in the sample to be enhanced, thereby producing a composition of neuronal progenitor cells.

[0065] The first step of this method may involve harvesting a skin sample containing hair follicle cells. It is preferable to obtain the sample from a human scalp containing the highest and most uniform hair follicle density, preferably from the midline occipital scalp. This skin generally contains more dense active hair follicles depending on the individual. We found that skin areas containing more dense active hair follicles enable the production of a better quantity and quality of cells by this method compared to skin areas containing less dense hair follicles.

[0066] Hair follicle cells generally include progenitor cells and stem cells from different niches, including the bulge area, germ zone, and dermal papillae, which are collectively referred to as hair follicle progenitor cells. Hair follicle progenitor cells can express neuroectodermal biomarkers.

[0067] The harvested skin samples are then treated under conditions where a significant number of hair follicle progenitor cells can transition to the growth phase. This step is very important because it contributes to an increased yield of neural progenitor cells. An example of this step is described in detail in the embodiments presented here.

[0068] This step typically involves the in vitro conditioning of human skin samples, the goal of which is to provide conditions that support cell viability of the skin sample cells and enable them to transition to the growth phase (or the active growth phase of the hair follicle cell cycle).

[0069] Prior to this stage, the cells in the skin sample may be in the growth phase (anagen), resting phase (telogen), or any other phase of the hair follicle cycle. While not strictly necessary, it is desirable for most hair follicle progenitor cells to be in the growth phase at the time of harvesting. This is because the final result of in vitro culture is that most hair follicle progenitor cells transition to the growth phase.

[0070] Anti-refractory hair follicle factors and / or pro-growth factors can be used to promote the transition of hair follicle progenitor cells and stem cells to the anagen phase, thereby enabling hair follicle cells in the skin to transition to the anagen phase. Examples of anti-refractory hair follicle factors include noggin or sonic hedgehog (SHH), factors that activate the Wnt1 signaling pathway, or factors that inhibit the stimulation of bone morphogenetic proteins (pro-refractory). Other examples of anti-refractory hair follicle factors include WNTs as well as BMP antagonists such as Grem 1 and Bambi. TGF-β2 is a major pro-growth factor, and other pro-growth factors include FGF7, FGF10, and platelet-derived growth factor (PDGF).

[0071] In vitro conditioning of skin samples generally uses culture media that support in vitro epidermal cell viability and hair follicle organogenesis. Williams medium E is one example. Other examples include different combinations of Dulbecco's modified eagle medium (DMEM) plus F-12, different combinations of F12 plus mammalian serum, and differently supplemented phosphate-buffered saline combinations.

[0072] In some cases, longer incubation may be possible, but generally, in vitro conditioning is performed for 12 to 100 hours.

[0073] It is possible to monitor the in vitro culture process during the culture period. For example, the characteristics of the cells or culture can be evaluated by the growth of hair stems within isolated hair follicles.

[0074] When in vitro cell culture is completed, it is desirable that at least 80% of the cells are in the growth phase (anagen phase).

[0075] At the completion of culture in which the conditioned skin sample is obtained, many cells will remain entrapped within the conditioned skin by the extracellular matrix and non-cellular components of the skin tissue. These cells will be released from the skin tissue. Depending on the method, cells are released from the conditioned skin by contacting the skin with one or more enzymes under conditions capable of degrading the extracellular matrix of the conditioned skin, thereby releasing the cells into the sample. Generally, the enzymes are one or more selected from the group consisting of trypsin, DNase, dispase, collagenase, and combinations of accustase and TrypLE.

[0076] Depending on the properties of the conditioned skin, on the other hand, it may also be possible to release cells from the conditioned skin by mechanical means that divide the conditioned tissue into smaller pieces of tissue or separate them.

[0077] In some embodiments, cells may be released from skin conditioned by a combination of enzymatic or mechanical treatment. Generally, the enzymatic treatment is initiated before the mechanical treatment, but the enzymatic or mechanical treatments may occur simultaneously.

[0078] When the step of releasing cells from a conditioned skin tissue sample is completed, the non-cellular components of the released cells and extracellular matrix and the non-cellular components of the skin tissue are provided, which can be suspended in a cell medium. These are generally removed before the start of a subsequent step. Centrifugation and filtration may be used. An example of this method is described in the example herein.

[0079] The composition of cells released from conditioned skin is heterogeneous and, depending on where the tissue is harvested, includes neuronal progenitor cells, other multifunctional cells, and finally differentiated cells, including fibroblasts, keratinocytes, and other cells of the epithelium and skin layers of the skin tissue. The next step requires obtaining a cell sample that contains a majority of neuronal progenitor cells. This then requires the removal of non-neuronal progenitor cells from the composition of cells released from conditioned human skin cells. The cells to be removed are typically finally differentiated cells (i.e., keratinocytes, fibroblasts, other skin and epithelial cells, and apoptotic cells).

[0080] In one embodiment, finally differentiated cells are depleted from the sample by contacting the sample with a reagent that selectively depletes the finally differentiated cells from the sample under conditions that selectively deplete the finally differentiated cells from the sample. Preferably, the reagent is an antibody that binds to finally differentiated cells but not to finally undifferentiated cells. An antibody that is a monoclonal antibody that does not bind to neuronal progenitor cells is preferably effective at this step. Preferably, the antibody binds to epithelial or skin cells such as keratinocytes or fibroblasts. Examples of antibodies include those that selectively bind to fibroblast-specific antigen 1 or CD45, which are not antigens found on the surface of neuronal progenitor cells.

[0081] When this depletion phase is complete, the sample contains less than 5% of finally differentiated cells.

[0082] Therefore, according to the first embodiment of the present invention, a method for producing a composition of neuronal progenitor cells or a composition of growth-capable cells expressing a neuronal lineage biomarker is provided, comprising:

[0083] - A skin sample, which is skin containing hair follicle cells, is treated in a condition in which the hair follicle cells in the skin can transition to the growth phase, thereby forming a conditioned skin sample;

[0084] - Release cells as a sample from the conditioned skin;

[0085] - Finally, deplete differentiated cells, dead cells, or cell debris from the sample, thereby forming a sample of finally undifferentiated cells; and optionally

[0086] - Treat a sample of finally undifferentiated cells under conditions that allow the number of finally undifferentiated cells in the sample to expand;

[0087] Thereby, it includes the production of a composition of neuronal progenitor cells.

[0088] This method may be performed without a separate step of releasing cells from conditioned skin. For example, cells may be released from the skin into a sample during the condition of the treatment step in which hair follicle cells transition to the growth phase. Therefore, this method may widely involve the production of a composition of proliferative cells that are enhanced with neuronal progenitor cells or express neuronal lineage biomarkers, which;

[0089] - A skin sample, which is skin containing hair follicle cells, is treated in a condition in which the hair follicle cells in the skin can transition to the growth phase, thereby forming a conditioned skin sample;

[0090] - Finally, deplete the differentiated cells, dead cells, or cell debris from the conditioned sample, thereby forming a sample of finally undifferentiated cells;

[0091] - By treating the sample with finally undifferentiated cells under conditions that allow the number of cells containing neuronal lineage biomarkers in the sample to be enhanced;

[0092] This enables the production of a composition of proliferative cells that express neuronal progenitor cells or neuronal lineage biomarkers.

[0093] Otherwise, it may be necessary to perform a separate process step to release cells from the conditioned skin, and the method includes:

[0094] - A skin sample, which is skin containing hair follicle cells, is treated in a condition in which the hair follicle cells in the skin can transition to the growth phase, thereby forming a sample of the conditioned skin;

[0095] - Release cells as a sample from the conditioned skin;

[0096] - Finally, deplete differentiated cells, dead cells, or cell debris from the conditioned skin sample, thereby forming a sample of finally undifferentiated cells;

[0097] - Treat a sample of finally undifferentiated cells under conditions that allow the number of cells containing neuronal lineage biomarkers in the sample to be enhanced;

[0098] This is equivalent to producing a composition of proliferative cells that express neuronal progenitor cells or neuronal lineage biomarkers.

[0099] In some embodiments, the step of depleting finally differentiated cells from a conditioned skin sample may be performed during the treatment of a sample of non-terminally differentiated cells in a condition that allows the number of cells containing neuronal lineage biomarkers to be enhanced—or as part of this step. For example, in one embodiment, the condition resulting in an enhancement of the number of cells containing neuronal lineage biomarkers may include a condition in which the loss of cells likely to develop into other cells rather than finally differentiated cells, pluripotent cells, or neuronal lineage cells is more favorable. Therefore, in one embodiment, the method is:

[0100] - A skin sample, which is skin containing hair follicle cells, is treated in a condition in which the hair follicle cells in the skin can transition to the growth phase, thereby forming a sample of the conditioned skin;

[0101] - Optionally, release cells into a sample from the conditioned skin;

[0102] - Treat a sample of cells from skin conditioned in a condition that allows the number of cells containing neuronal lineage biomarkers in the sample to be enhanced;

[0103] This may include a method for producing a composition of proliferable cells that express neuronal progenitor cells or neuronal lineage biomarkers.

[0104] A second embodiment of the present invention is described herein, wherein hair follicle samples, separated from the skin tissue attached to them in their natural state or otherwise separated so as not to be attached to the skin tissue, are used to induce a composition of neuronal progenitor cells. A particular advantage of using separated hair follicles is that this method can be carried out without the substantial use of enzymatic or mechanical means that would otherwise require the release of hair follicle cells from the surrounding skin tissue. Furthermore, this also avoids contamination by ultimately differentiated skin cells and non-neuronal lineage cells in subsequent cultures. According to the embodiment, this method produces a composition enriched with neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers, and comprises the following steps:

[0105] - A sample of hair follicle cells is treated under conditions where the hair follicle cells can transition to the growth phase, thereby forming a sample of conditioned cells;

[0106] - Treat a sample of conditioned cells in a condition where the formation of neural bulbs from the cells of the conditioned cell sample is possible;

[0107] This produces proliferative cells that express neuronal progenitor cells or neuronal lineage biomarkers.

[0108] Conditioned cell samples generally contain hair follicle cells in the growth phase that are enhanced and cells showing neuronal lineage biomarkers.

[0109] When the formation of neurospheres is complete, it would be advantageous to increase the number of neurosphere cells, which are neuronal progenitor cells, or cells expressing neuronal lineage biomarkers by means of an expansion step. Various known techniques exist for this purpose. Therefore, in another embodiment, a method is provided for producing a composition enhanced with neuronal progenitor cells or proliferable cells expressing neuronal lineage biomarkers, the method comprising:

[0110] - A sample of hair follicle cells is treated under conditions where the hair follicle cells can transition to the growth phase, thereby forming a sample of conditioned cells;

[0111] - Treat a sample of conditioned cells under a condition that enables the formation of a neuroglobule sample from the cells of the conditioned cell sample;

[0112] - Provides a condition in which the number of neuroglobules in the neuroglobule sample expands;

[0113] This includes the production of a composition of proliferable cells expressing neuronal progenitor cells or neuronal lineage biomarkers.

[0114] When the treatment step of transferring hair follicle cells to a growth phase is completed, the cells may clump or group together with other cells or materials, in which case the cells may be separated to form a single-cell suspension. This can be achieved by many techniques known in the art. In one embodiment, enzymatic separation using an enzyme useful for separating hair follicle cells into single cells is used. Therefore, in another embodiment, a method is provided for producing a composition enhanced with neuronal progenitor cells, or proliferable cells expressing neuronal lineage biomarkers, the method comprising:

[0115] - A sample of hair follicle cells is treated under conditions where the hair follicle cells can transition to the growth phase, thereby forming a sample of conditioned cells;

[0116] - Provides a condition in which a sample of conditioned cells can be separated into single cells by enabling cell separation;

[0117] - Treat a sample of conditioned cells under a condition that enables the formation of a neuroglobule sample from the cells of the conditioned cell sample;

[0118] - Provides a condition in which the number of neuroglobules in the neuroglobule sample expands;

[0119] This includes the production of a composition of proliferable cells expressing neuronal progenitor cells or neuronal lineage biomarkers.

[0120] According to a second embodiment of the present invention, approximately 200 hair follicle samples obtained from the midline occipital scalp can be subjected to conditions in a pro-anagenic environment for a period of up to 100 hours. The resulting cells can be enzymatically separated into single cells, 10 5 It is provided in the form of cells. These cells are then 10 under conditions where neuroglobules can form. 4It can be cultured in a suspension diluted to a cell / ml level. This suspension is then filtered to harvest only the neuroglobules, the cells are isolated by enzymatic treatment, and attached and expanded into a monolayer.

[0121] It will be understood that the invention disclosed and defined in this specification applies to all or more different combinations of two or more of the individual features mentioned. All of these different combinations constitute various other aspects of the invention.

[0123] Implementation example

[0124] Implementation Example 1 - Harvest of donor tissue

[0125] Human adult skin was harvested from the occipital scalp as follows: prior to harvesting, the occipital bone was shaved, sterilized, and anesthetized. Using dual-blade scalpels, a 10 mm wide, 3 cm long sample was obtained along the lateral circumference around the midline, with the thickness extending to the underlying fat layer.

[0127] Example 2 - Incubation of donor tissue with hair cycle regulatory factors

[0128] Conditioning donor skin tissue with anti-refractory or pro-proliferative hair follicle regulators for up to 100 hours increases the anage:telogen ratio across skin samples as well as inter-follicular synchrony. Pre-treatment of donor skin tissue with such factors therefore increases terminal cell viability, yield, and uniformity. Skin culture is performed in an environment of supplemented Williams' E media, which provides increased hair follicle viability in vitro. Noggin or SHH, as examples of anti-refractory hair follicle cell cycle factors, increases the anage:telogen ratio and thus increases terminal cell viability, yield, and uniformity. TGF-β2, as a proliferative example of a hair follicle cell cycle factor, increases the growth-to-resting-phase ratio and thus increases terminal cell viability, yield, and uniformity. The combination of Noggin, SHH, and TGF-β2 in supplemented Williams' E media provides high cell viability, yield, and uniformity. Indeed:

[0129] 1. Excess subcutaneous fat is trimmed and removed so that the skin protrusions appear as dark spots and are kept on the fat side of the skin sample.

[0130] 2. Go ahead and wash the remaining skin 3 times with 1% anti-anti in PBS, and

[0131] 3. The sample was cut into 4mm x 10mm pieces with a surgical knife.

[0132] 4. Skin fragments were placed in William's E medium supplemented (containing 11.1 mM glucose and 2 mM L-glutamine) with 10 micrograms / ml insulin plus: 100-500 ng / ml Nogyn ( noggin (12-100 hours), 100-500ng / ml SHH (12-100 hours), or 50ng-200ml TGF-β2 (Foitzik et al., 1999a) Incubate in a medium supplemented with (12-100 hours), or a combination thereof.

[0134] Example 3 - Mechanical processing of skin and treatment with digestive enzymatic agent.

[0135] Skin separation via a mechanical device combined with enzymatic digestion for the release of progenitor cells from the extracellular matrix can increase cell viability, yield, and uniformity. An exemplary combination is the use of the Miltenyi Biotec gentleMACS Dissociator device with the gentleMACS enzymatic digestion kit, which together increases cell viability, yield, and uniformity.

[0136] In addition to the proprietary description of use, the customized approach to cell filtration provides high viability, yield, and uniformity.

[0137] 1. Prepare the GentleMACS dissociation enzyme (the following volume is for a 4 x 4 mm skin piece).

[0138] 2. Carefully mix Buffer L (435 µl) and Enzyme P (12.5 µl).

[0139] 3. Carefully mix Enzyme D (50 ul) and Enzyme A (2.5 ul).

[0140] 4. Add the D / A mixture to the L / P mixture in the C-tube.

[0141] 5. Place 4 pieces of skin into the C-tube containing the enzyme and twist the lid shut.

[0142] 6. Incubate at 37°C for 12 hours.

[0143] The next day

[0144] 7. Before starting, prepare at least 50 mL of cold wash media (DMEM / F12 3:1 + 1% anti-anti) and warm at least 50 mL of growth media (DMEM / F12 3:1 20 ng / mL EGF + 40 ng / mL FGF2 + 2% B27).

[0145] 8. Dilute the contents of each C-tube by adding 0.5 mL of cold wash medium, and

[0146] 9. Close the C-tube lid tightly and attach this tube upside down to the sleeve of the GentleMACS Dissociator.

[0147] 10. Select the h_skin_01 program in GentleMACS and press Start.

[0148] 11. Centrifuge the contents of the tube at 300xG for 45 seconds and collect them at the bottom.

[0149] 12. Empty the contents from the C-tube into the wire mesh strainer on the 6-well plate well(s) and

[0150] 13. Rinse the contents from a sieve into the wells using a washing medium, and

[0151] 14. As you proceed, remove any large, obstructive tissue fragments from the sieve. Note: Remove only the tissue fragments after thoroughly washing with pipetting in cold medium.

[0152] 15. Thoroughly rinse all C-tubes with a 50 mL tube using washing medium.

[0153] 16. Pipette this from the sieve into the same 6-well(s) and

[0154] 17. Place a 40uM sieve on a 50mL tube, and moisten the sieve mesh with washing medium.

[0155] 18. Filter the cell / medium mixture through a 40 µM sieve and

[0156] 19. Filter the cell / medium mixture through a second 40 µM sieve and repeat the previous filtration step.

[0157] 20. Centrifuge the cells at 300°G for 10 minutes. Completely remove the supernatant.

[0159] Implementation Example 4 - Depletion of potential contaminatory cells

[0160] Depleting potentially contaminated cells increases cell viability, yield, and uniformity compared to not performing such depletion. An exemplary approach is to use the Miltenyi MACS magnetic cell isolation system for the depletion of such living cells. Using this device, the depletion of ultimately differentiated cell types increases cell viability, yield, and consistency. One or more markers are: fibroblasts (( fibroblast-specific antigen 1 )) or epithelial cells (epithelial cells) CD45 It can be used for the depletion of mature, non-neuroglobinogenic cells such as ). Using this device to deplete dead cells and debris with the Miltenyi Dead Cell Removal Kit also increases cell viability, yield, and consistency.

[0161] For the depletion of dead cells:

[0162] 1. 107 of Dilute 0.25 mL of 20X binding buffer stock solution per total cell with 4.75 mL of sterile double-distilled water.

[0163] 2. The cell pellet is about 10 7 of Resuspend in 100 μL of Dead Cell Removal MicroBeads per total cell.

[0164] 3. Mix well and incubate at room temperature (20-25 °C) for 15 minutes.

[0165] 4. Select a column type according to the number of cells and place the column in the magnetic field of a MACS separator.

[0166] 5. Prepare the column by rinsing with 1X binding buffer (MS: 500 μL; LS: 3 mL).

[0167] 6. Apply an appropriate amount of cell suspension in 1X binding buffer to the column (MS: 500-1000 μL; LS: 1-10 mL). Pass the negative (unlabeled) cell fraction into a 15 mL tube containing warmed DMEM.

[0168] 7. Rinse the column with an appropriate amount of 1X binding buffer (MS: 4×500 μL; LS: 4×3 mL) and collect unlabeled cells.

[0169] 8. Centrifuge the cells at 300°G for 10 minutes.

[0170] 9. Prepare a buffer by diluting the MACS BSA stock solution with autoMACS rinsing solution at a ratio of 1:20. Keep the buffer cold (2-8 °C).

[0171] 10. Completely remove the supernatant from the cells and the cell pellet 10 7 of Resuspend in 80 μL of buffer per cell. 10 7 of Add 20 μL of microbeads (e.g., anti-fibroblast beads) per cell.

[0172] 11. Mix well and incubate at room temperature (19-25 °C) for 30 minutes.

[0173] 12. 10 7 Wash the cells by adding 1-2 mL of buffer per cell and centrifuge at 300 KJ for 10 minutes.

[0174] 13. Completely aspirate the supernatant. 10 in 500 μL of buffer 8 Resuspends down to the cells,

[0175] 14. Select the column type according to the number of cells and place the column in the magnetic field of the MACS separator. Rinse with buffer to prepare the column.

[0176] 15. Apply the cell suspension to the column. Collect the flow-through containing unlabeled cells. Wash the column with an appropriate amount of buffer. Collect the flow-through unlabeled cells and mix them with the effluent cell suspension.

[0178] Example 5 - 3D Neurosphere Formation

[0179] 1. Centrifuge the unlabeled cell fraction at 350xG for 10 minutes.

[0180] 2. Remove the supernatant and resuspend the pellet in 5 mL of SKN growth medium (DMEM / F12 (3:1), 20 ng / mL EGF, 40 ng / mL bFGF, and 2% B27).

[0181] 3. Remove 10 µL and transfer to a microvial, then mix with 10 µL of Trypan blue. Record the cell count and viability.

[0182] 4. Dilute the cell suspension with SKN growth medium and place 1 x 10⁶ cells in 6 mL per well of a non-tissue culture-treated 6-well plate. 6 Inoculate with cells. Record the number of inoculated wells and the exact density.

[0183] 5. Incubate at 37°C in 5% CO2 - this indicates 0 days of incubation.

[0184] 6. Move all records and images to the archive.

[0185] 7. Day 1 of culture: Evaluate for signs of contamination and discard the culture if necessary.

[0186] 8. Days 5 and 7 of culture: Take photos of the cells at low and high magnification (mag) and measure the size of the neuroglobules. Record observations regarding culture growth. If most neuroglobules have a diameter >50 µm (approximately 1 day after first appearing), proceed with 2D monolayer expansion. After 7 days, monitor neuroglobule size daily. Most neuroglobules should be detached before they become >100 µm or before they attach.

[0188] Example 6 - 2D Monolayer Expansion

[0189] Avoid allowing spheres to begin adhering to the plate, as this will make them difficult to recover and separate. The goal is for most spheres to have a diameter of approximately 100 μm. Large, irregular cell clumps appearing during the first 1-2 days are cell aggreates, not neurospheres. These indicate that the cell density is too high or that the tissue has not been sufficiently separated.

[0190] 1. Before starting, cover a T25 flask (or a large amount is required) with 0.5 μg / cm2 Laminin 511 (Laminin 511) and polymerize overnight at 4°C, and warm 7-14 mL of growth medium (7 mL is needed per T25 cell) - DMEM / F12 3:1 with 1% pen-strep, 20 ng / mL EGF, 40 ng / mL bFGF and 2% B27, and dissolve 1 mL of TrypLE at 4°C.

[0191] 2. Take photos of the sphere under both high and low magnification.

[0192] 3. Transfer all culture medium containing spheres from the culture well to a centrifuge tube.

[0193] 4. Centrifuge at 300xG for 10 minutes.

[0194] 5. Remove the supernatant and resuspend in 1 mL of TrypLE. Incubate for 5 minutes.

[0195] 6. Remove the cells from the incubator and detach the sphere by pipetting up and down 100-200 times with a pipette (using a 200 uL pipette).

[0196] 7. Add 2 mL of growth medium and mix well.

[0197] 8. Remove 10 uL of the suspension, place it in a small vial with 10 uL of Trypan blue, and count the number of cells.

[0198] 9. Record the total number of surviving and dead cells.

[0199] 10. Dilute the cell suspension further in warm growth medium as needed.

[0200] 11. To prepare the T25 flask, remove Laminin 511.

[0201] 12. 1x10 4 Cells / cm2 (=2.5x10 5 Inoculate 7 mL of cells (per T25 flask). This is PO.

[0202] 13. Return the cells to the incubator with minimal movement for the first 24 hours.

[0203] 14. Replace the medium with fresh growth medium after 24 hours.

[0204] 15. Change the growth medium every 3 days. When the flask is fused to ~80%, subculture the cells with TryPLE to obtain 1 x 10⁶ 4 cells / cm 2 Re-vaccinate.

[0206] References

[0207] BELICCHI, M., PISATI, F., LOPA, R., PORRETTI, L., FORTUNATO, F., SIRONI, M., SCALAMOGNA, M., PARATI, EA, BRESOLIN, N. & TORRENTE, Y. 2004. Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain. J Neurosci Res, 77, 475-86.

[0208] BIERNASKIE, JA, MCKENZIE, IA, TOMA, JG & MILLER, FD 2007. Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny. Nature Protocols, 1, 2803.

[0209] FOITZIK, K., PAUS, R., DOETSCHMAN, T. & DOTTO, G. P. 1999a. The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis. Dev Biol, 212, 278-89.

[0210] FUCHS, E. 2018. Skin Stem Cells in Silence, Action, and Cancer. Stem Cell Reports, 10, 1432-1438.

[0211] GE, Y., GOMEZ, N. C., ADAM, R. C., NIKOLOVA, M., YANG, H., VERMA, A., LU, C. P.-J., POLAK, L., YUAN, S., ELEMENTO, O. & FUCHS, E. 2017. Stem Cell Lineage Infidelity Drives Wound Repair and Cancer. Cell, 169, 636-650.e14.

[0212] HWANG, I., CHOI, K. A., PARK, H. S., JEONG, H., KIM, J. O., SEOL, K. C., KWON, H. J., PARK, I. H. & HONG, S. 2016. Neural Stem Cells Restore Hair Growth Through Activation of the Hair Follicle Niche. Cell Transplant, 25, 1439-51.

[0213] JOANNIDES, A., GAUGHWIN, P., SCHWIENING, C., MAJED, H., STERLING, J., COMPSTON, A. & CHANDRAN, S. 2004. Efficient generation of neural precursors from adult human skin: astrocytes promote neurogenesis from skin-derived stem cells. The Lancet, 364, 172-178.

[0214] PANG, Z. P., YANG, N., VIERBUCHEN, T., OSTERMEIER, A., FUENTES, D. R., YANG, T. Q., CITRI, A., SEBASTIANO, V., MARRO, S., SUDHOF, T. C. & WERNIG, M. 2011. Induction of human neuronal cells by defined transcription factors. Nature, 476, 220-3.

[0215] SON, E. Y., ICHIDA, J. K., WAINGER, B. J., TOMA, J. S., RAFUSE, V. F., WOOLF, C. J. & EGGAN, K. 2011. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell, 9, 205-18.

[0216] TOMA, J. G., AKHAVAN, M., FERNANDES, K. J. L., BARNABE-HEIDER, F., SADIKOT, A., KAPLAN, D. R. & MILLER, F. D. 2001. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol, 3, 778-784.

[0217] TOMA, J. G., MCKENZIE, I. A., BAGLI, D. & MILLER, F. D. 2005. Isolation and characterization of multipotent skin-derived precursors from human skin. Stem Cells, 23, 727-37.

[0218] TRUONG, A., SI, E. S., DUNCAN, T. & VALENZUELA, M. 2016. Modeling neurodegenerative disorders in adult somatic cells: A critical review. Front. Biol., 11, 232-245.

[0219] TSUNEMOTO, R., LEE, S., SZUCS, A., CHUBUKOV, P., SOKOLOVA, I., BLANCHARD, J. W., EADE, K. T., BRUGGEMANN, J., WU, C., TORKAMANI, A., SANNA, P. P. & BALDWIN, K. K. 2018. Diverse reprogramming codes for neuronal identity. Nature.

[0220] VALENZUELA, M. J., DEAN, S. K., SACHDEV, P., TUCH, B. E. & SIDHU, K. S. 2008. Neural precursors from canine skin: a new direction for testing autologous cell replacement in the brain. Stem Cells Dev, 17, 1087-94.

[0221] VIERBUCHEN, T., OSTERMEIER, A., PANG, Z. P., KOKUBU, Y., SUDHOF, T. C. & WERNIG, M. 2010. Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 463, 1035-1041.

[0222] YANG, H., ADAM, R. C., GE, Y., HUA, Z. L. & FUCHS, E. 2017. Epithelial-Mesenchymal Micro-niches Govern Stem Cell Lineage Choices. Cell, 169, 483-496.e13.

[0223] YU, H., FANG, D., KUMAR, S. M., LI, L., NGUYEN, T. K., ACS, G., HERLYN, M. & XU, X. 2006. Isolation of a Novel Population of Multipotent Adult Stem Cells from Human Hair Follicles. The American Journal of Pathology, 168, 1879-1888.

[0224] YU, H., KUMAR, S. M., KOSSENKOV, A. V., SHOWE, L. & XU, X. 2010. Stem Cells with Neural Crest Characteristics Derived from the Bulge Region of Cultured Human Hair Follicles. Journal of Investigative Dermatology, 130, 1227-1236.

[0225] ZHOU, D., ZHANG, Z., HE, L. M., DU, J., ZHANG, F., SUN, C. K., ZHOU, Y., WANG, X. W., LIN, G., SONG, K. M., WU, L. G. & YANG, Q. 2014. Conversion of fibroblasts to neural cells by p53 depletion. Cell Rep, 9, 2034-42.

Claims

Claim 1 A method for producing a composition comprising at least one of pluripotent neuronal precursor cells and proliferable pluripotent cells expressing neural lineage biomarkers, comprising: treating a hair follicle sample with one or more factors to promote the transition of hair follicle cells from the telogen phase to the anagen phase, thereby forming a conditioned cell sample; providing conditions so that the conditioned cell sample can be dissociated into single cells; treating the cells with conditions so that a neuroglobule sample can be formed from the cells; providing conditions to the neuroglobule sample so that the number of cells in the neuroglobule can be expanded; thereby producing one or more of pluripotent neuronal precursor cells and proliferable pluripotent cells expressing neural lineage biomarkers. Claim 2 A method according to claim 1, wherein the hair follicle sample comprises an intact hair follicle containing hair follicular precursor cells. Claim 3 A method according to paragraph 2, wherein the subpopulation of hair follicle progenitor cells expresses neuro-ectodermal biomarkers. Claim 4 A method according to any one of claims 1 to 3, wherein the hair follicle cells are provided as a sample of skin. Claim 5 The method of claim 1, wherein one or more factors are selected from factors activating the Wnt signaling pathway, TGF-β, inhibitors or antagonists of bone morphogenic proteins, and pro-growth factors. Claim 6 In claim 5, the method wherein the factor activating the Wnt signal transmission path is WNT, Noggin, or Sonic Hedgehog (SHH). Claim 7 In paragraph 5, the method wherein the inhibitor or antagonist of the bone-forming protein is BMP, Grem1, or Bambi. Claim 8 In claim 5, the method wherein the pro-growth factor is FGF7, FGF10, or platelet-derived growth factor (PDGF). Claim 9 In paragraph 6, the above factor is a noggin or a sonic hedgehog (SHH), a method. Claim 10 In paragraph 5, the above-mentioned growth factor is TGF-β2, method. Claim 11 A method according to claim 1, wherein the hair follicle sample is cultured in a culture medium suitable for maintaining hair follicle cells. Claim 12 In paragraph 11, the method wherein the above-mentioned medium is Williams medium E. Claim 13 In paragraph 4, the method wherein the skin is human skin or the hair follicle is a human hair follicle. Claim 14 In paragraph 4, the method wherein the skin or hair follicle is obtained from a human scalp. Claim 15 In paragraph 4, the method wherein the skin or hair follicle is the midline occipital scalp. Claim 16 In claim 1, the hair follicle sample comprises a skin sample, and the method comprises: treating the skin sample in a condition in which hair follicle cells within the sample can transition from the telogen phase to the anagen phase, thereby forming a conditioned skin sample; releasing cells from the conditioned skin into the sample; depleting the finally differentiated cells, or the dead cells, or cell debris from the sample, thereby forming a sample of non-terminally differentiated cells; and treating the sample of non-terminally differentiated cells in a condition in which the number of non-terminally differentiated cells can expand, thereby producing a composition comprising at least one of pluripotent neural progenitor cells and proliferable pluripotent cells expressing a neuronal lineage biomarker. Claim 17 In claim 16, a method of contacting the skin with one or more enzymes in a condition in which the extracellular matrix of the skin, conditioned to release cells into a sample, can be degraded, thereby causing the cells to be released from the skin. Claim 18 In claim 17, the enzyme is selected from the group consisting of trypsin, DNase, dispase, and collagenase. Claim 19 In claim 16, a method of releasing cells from the conditioned skin by mechanically destroying the extracellular matrix of the conditioned skin to release the cells as a sample. Claim 20 In paragraph 19, the method wherein the mechanical destruction includes manual trituration. Claim 21 A method according to claim 16, comprising the step of removing non-cellular components after releasing cells from the conditioned skin into a sample and before finally depleting the differentiated cells from the sample. Claim 22 In claim 16, a method of depleting the finally differentiated cells from a sample by contacting the sample with a reagent under conditions that enable the selective depletion of the finally differentiated cells from the sample. Claim 23 In paragraph 22, the method wherein the reagent is an antibody that binds to finally differentiated cells but not to finally undifferentiated cells. Claim 24 In paragraph 23, the method wherein the reagent is an antibody that does not bind to neural progenitor cells. Claim 25 In claim 22, the method wherein the antibody is an antibody that binds to epidermal or dermal cells, or binds to epidermal cells, keratinocytes, or fibroblasts, or binds to fibroblast-specific antigen 1 or CD45, or binds to all of these. Claim 26 In paragraph 16, a method in which, when the depletion step is completed, the sample ultimately contains less than about 5% of differentiated cells. Claim 27 In claim 1, the composition produced by the above method has the following characteristics: <5% of the cells express astroglial GFAP, adiponectin, oligodendrocyte O4, and myofibroblast SMA, and >90% of the cells express nestin, CD133 and III-tubulin ( A method that possesses the characteristic of being positive for III-tubulin. Claim 28 The method according to claim 1, wherein the composition produced by the above method has a neuronal yield of 90 to 100% after neuronal differentiation in vitro. Claim 29 In claim 1, the cells of the composition are 10 within 4 weeks 7 dog At the level A method that can be expanded to produce homogeneous and single-function neural progenitor cells. Claim 30 A method according to claim 1, wherein the conditions for expanding the number of cells in the neuroglobules include harvesting the neuroglobules, isolating the cells, and culturing the isolated cells in an adherent monolayer culture to expand the number of cells in the neuroglobules.

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