Treatment of nerve regeneration-promoting cells (NRPCs) and damaged nerve cells

Abstract

This disclosure provides novel and innovative compositions of neuronal regeneration-promoting cells (NRPCs), methods for generating NRPCs, and methods for treating subjects having damaged nerve cells using NRPCs. In one embodiment, the NRPCs are derived from tonsil-derived mesenchymal stem cells and express CD26, CD106, CD112, CD121a, and CD141, where CD121a has an expression level of approximately 30% or higher, and this expression level is measured immediately after thawing the NRPCs from a thawed state. Furthermore, the NRPCs are configured to promote axon formation on neuronal cells.

Description

[Technical Field]

[0001] This disclosure relates to stem cell-derived neuronal regeneration-promoting cells (NRPCs), and more specifically, to NRPCs and methods thereof that have the ability to treat damaged nerve cells. [Background technology]

[0002] Neurons, or nerve cells, form the building blocks of the nervous system, converting and relaying electrical signals. A neuron may include a cell body, dendrites extending from the cell body, and an axon. The axon is the long output structure of a neuron, enabling it to propagate electrical signals as action potentials. Some axons are encased in a fatty substance called myelin, which allows electrical signals to propagate more efficiently through the neuron. Myelin acts as an insulator for the axon, helping to transmit its signals over long distances. The development of myelin around the axon, or myelin sheathing, can be promoted by nerve regeneration-promoting cells (NRPCs).

[0003] Various neurological disorders can result from inadequate or insufficient myelination of nerve cells. For example, Charcot-Marie-Tooth (CMT) disease is a hereditary disorder affecting 1 in 2,500 people, with heterogeneous phenotypes and genetic causes. CMT1A (CMT1A) is a type of hereditary neurological disorder that affects peripheral nerves and is caused by duplication of the peripheral myelin protein 22 (PMP22) gene. Therefore, effective treatments for CMT, including CMT1A, that can promote myelination while regulating PMP22 overexpression are desired and needed.

[0004] Mesenchymal stem cells (MSCs) are widely used in the development of cell therapies because they can differentiate into various cell types in response to specific stimuli. However, the ability of MSCs to effectively develop precursor cells for myelinating neurons is hampered by various factors, including low yields or insufficient neurite growth. Therefore, there is a demand and need for more effective NRPCs and methods for generating them.

[0005] Various embodiments that address one or more of these drawbacks are presented herein. [Overview of the Initiative]

[0006] The inventors have found that conventional methods for preparing nerve (neuron) regeneration-promoting cells (NRPCs) from stem cells are extremely difficult, and therefore the resulting NRPCs may not be very effective in treating damaged nerve cells. This disclosure provides novel and innovative compositions of NRPCs, methods for generating NRPCs, and methods for treating subjects having damaged nerve cells using NRPCs.

[0007] Therefore, in general embodiments, this disclosure provides a composition of nerve regeneration promoting cells (NRPCs). NRPCs are derived from tonsil-derived mesenchymal stem cells. NRPCs express CD26, CD106, CD112, CD121a, and CD141. The expression level of CD121a is ≥30%.

[0008] In some embodiments of this disclosure, which may be combined with any other embodiments, the CD121a expression level in NRPC is approximately 30% to approximately 50%, and the CD121a expression level is measured immediately after thawing the NRPC from a frozen state.

[0009] In some embodiments of this disclosure, which may be combined with other embodiments, the expression level of any given protein (e.g., CD121a) is said to be measured immediately after thawing the NRPC from a frozen state, if the expression level is measured before any of the one or more subsequent passages after thawing the NRPC from a frozen state.

[0010] In some embodiments of this disclosure, which may be combined with any other embodiments, the expression level of CD121a in NRPC is approximately 50% or higher, and the expression level of CD121a is measured after one or more passages of NRPC thawed from a frozen state.

[0011] In some embodiments of this disclosure, which may be combined with any other embodiments, the expression level of CD121a in NRPC is approximately 60% or higher, and the expression level of CD121a is measured after one or more passages of NRPC thawed from a frozen state.

[0012] In some embodiments of this disclosure, which may be combined with any other embodiments, the expression level of CD121a in NRPC is approximately 70% or higher, and the expression level of CD121a is measured after one or more passages of NRPC thawed from a frozen state.

[0013] In some embodiments of this disclosure, which may be combined with any other embodiments, the expression level of CD121a in NRPC is approximately 80% or higher, and the expression level of CD121a is measured after one or more passages of NRPC thawed from a frozen state.

[0014] In some embodiments of this disclosure, which may be combined with any other embodiments, the expression level of CD121a in NRPC is approximately 90% or higher, and the expression level of CD121a is measured after one or more passages of NRPC thawed from a frozen state.

[0015] In one aspect of this disclosure, which may be combined with any other aspect, the expression level of CD26 in NRPC is 5% or less, the expression level of CD106 in NRPC is 15% or more, the expression level of CD112 in NRPC is 50% or more, and the expression level of CD141 in NRPC is 30% or less. The expression levels of CD26, CD106, CD112, and CD141 are measured immediately after thawing the NRPC from a frozen state.

[0016] In one embodiment of this disclosure, which may be combined with any other embodiment, the expression level of CD26 in NRPC is ≥10%, the expression level of CD106 in NRPC is ≥10%, the expression level of CD112 in NRPC is ≥25%, and the expression level of CD141 in NRPC is ≥10%. The expression levels of CD26, CD106, CD112, and CD141 are measured after one or more passages of NRPC thawed from a frozen state.

[0017] In some embodiments of this disclosure, which may be combined with any other embodiments, the expression levels of CD26 in NRPCs are 10% to 35%, the expression levels of CD106 in NRPCs are 10% to 35%, the expression levels of CD112 in NRPCs are 25% to 90%, and / or the expression levels of CD141 in NRPCs are 10% to 45%. The expression levels of CD26, CD106, CD112, and CD141 are measured after one or more passages of NRPCs thawed from a frozen state.

[0018] In further embodiments, the Disclosure provides a method for producing NRPCs. This method includes: generating multiple cultures of tonsil-derived mesenchymal stem cells (tonsil-derived MSCs) to form neurospheres; generating multiple cell cultures from the neurospheres for induction into NRPC candidates; and selecting from the multiple NRPC candidates an NRPC that expresses CD26, CD106, CD112, CD121a, and CD141, and having a first expression level of about 30% or more for CD121a, wherein the first expression level is measured immediately after thawing the NRPC candidates from a frozen state.

[0019] In one embodiment of the present disclosure, which may be combined with any other embodiments, each of the plurality of cultures of tonsil-derived MSCs is produced in a separate container, each of which comprises a separate culture containing tonsil-derived MSCs and a culture medium for forming the neurospheres.

[0020] In certain aspects of the present disclosure, which may be combined with any other aspect, the method includes collecting neurospheres from each of at least a portion of a container containing neurospheres; and processing the collected neurospheres to further collect cells from the neurospheres.

[0021] In certain aspects of the present disclosure, which may be combined with any other aspect, each of the plurality of cultures derived from neurospheres is in a separate container, and each of the containers is generated such that it contains cells collected from the neurospheres and a culture medium for inducing the cells into NRPC candidates.

[0022] In certain aspects of the present disclosure, which may be combined with any other aspect, the left and right tonsillar tissues of a single individual provide two separate cultures of tonsil-derived MSCs.

[0023] In certain aspects of the present disclosure, which may be combined with any other aspect, the method further includes providing a plurality of cultures of tonsil-derived MSCs, which includes providing the left and right tonsillar tissues of a single human; isolating a first tonsil-derived MSC from the left tonsil; and isolating a second tonsil-derived MSC from the right tonsil.

[0024] In certain aspects of the present disclosure, which may be combined with any other aspect, the method further includes a selection that includes analyzing expression of CD markers.

[0025] In certain aspects of the present disclosure, which may be combined with any other aspect, the selection includes performing flow cytometry with respect to one or more of CD26, CD106, CD112, CD121a, or CD141.

[0026] In some embodiments of the present disclosure, which may be combined with any other embodiments, the method further comprises evaluating whether each of a plurality of NRPC candidates or a subset thereof induces myelination in the dorsal root ganglia, wherein the selection is made of NRPCs that induce myelination in the dorsal root ganglia, express CD26, CD106, CD112, CD121a, and CD141, and have a first expression level of CD121a of approximately 30% or higher, which is a first expression level measured immediately after thawing the NRPC candidate from a frozen state.

[0027] In some aspects of this disclosure, which may be combined with any other aspects, the evaluation includes: co-culturing and evaluating the dorsal root ganglion and the NRPC candidate under evaluation; and subsequently examining the dorsal root ganglion to confirm myelin formation.

[0028] In one embodiment of the present disclosure, which may be combined with any other embodiment, the method further comprises evaluating whether each of a plurality of NRPC candidates or a subset thereof induces neurite outgrowth on each sample of neuroblastoma cells, wherein a given NRPC candidate induces neurite outgrowth if the average number of neurites formed per neuroblastoma cell in each sample of neuroblastoma cells is at least 15 and the length of the longest neurite formed in each sample of neuroblastoma cells is at least 150 μm; wherein the selection further comprises selecting from the NRPC candidates an NRPC which induces neurite outgrowth and expresses the CD26, the CD106, the CD112, the CD121a, and the CD141, and for CD121a having the first expression level of about 30% or more, wherein the first expression level is measured immediately after the NRPC candidate is thawed from a frozen state.

[0029] In some embodiments of the present disclosure, which may be combined with any other embodiments, the method further includes: growing a selected NRPC over several passages; and recovering the NRPC from at least some of the passages.

[0030] In some embodiments of the present disclosure, which may be combined with any other embodiments, the method further includes discarding at least one NRPC candidate having a first expression level of less than 30% of CD121a.

[0031] In one embodiment of the present disclosure, which may be combined with any other embodiment, an NRPC is selected in which the second expression level of CD121a is approximately 75% or higher, and this second expression level is measured one or more passages after thawing the candidate NRPC from a frozen state.

[0032] In one embodiment of the present disclosure, which may be combined with any other embodiment, an NRPC is selected in which the second expression level of CD121a is approximately 80% or higher, and this second expression level is measured one or more passages after thawing the candidate NRPC from a frozen state.

[0033] In one embodiment of the present disclosure, which may be combined with any other embodiment, an NRPC is selected in which the second expression level of CD121a is approximately 85% or higher, and this second expression level is measured one or more passages after thawing the candidate NRPC from a frozen state.

[0034] In one embodiment of the present disclosure, which may be combined with any other embodiment, an NRPC is selected in which the second expression level of CD121a is approximately 90% or higher, and this second expression level is measured one or more passages after thawing the candidate NRPC from a frozen state.

[0035] In one embodiment of this disclosure, which may be combined with any other embodiment, an NRPC having a third expression level of approximately 5% or less for CD26 is selected; an NRPC having a fourth expression level of approximately 15% or more for CD106 is selected; an NRPC having a fifth expression level of approximately 50% or more for CD112 is selected; and an NRPC having a sixth expression level of approximately 30% or less for CD141 is selected. The third, fourth, fifth, and sixth expression levels are measured immediately after the NRPC candidate is thawed from a frozen state.

[0036] In one embodiment of this disclosure, which may be combined with any other embodiment, an NRPC having a seventh expression level of approximately 10% or more for CD26 is selected, an NRPC having a eighth expression level of approximately 10% or more for CD106 is selected, an NRPC having a ninth expression level of approximately 25% or more for CD112 is selected, and an NRPC having a tenth expression level of approximately 10% or more for CD141 is selected. The seventh, eighth, ninth, and tenth expression levels are measured after the NRPC candidate has been thawed from a frozen state and passed through one or more passages.

[0037] In one embodiment of this disclosure, which may be combined with any other embodiment, an NRPC having a seventh expression level of approximately 10% to approximately 35% for CD26 is selected; an NRPC having a eighth expression level of approximately 10% to approximately 35% for CD106 is selected; an NRPC having a ninth expression level of approximately 25% to approximately 90% for CD112 is selected; and an NRPC having a tenth expression level of approximately 10% to approximately 45% for CD141 is selected. The seventh, eighth, ninth, and tenth expression levels are measured after the NRPC candidate has been thawed from a frozen state and passed through one or more passages.

[0038] In further embodiments, the Disclosure provides a method for treating damaged nerve cells, comprising administering into the body of an object having damaged nerve cells an effective amount of any composition comprising the NRPC described herein to induce myelin formation of the damaged nerve cells or myelin regeneration of Schwann cells.

[0039] In further embodiments, the present disclosure provides a method for treating myofibrosis, comprising administering into the body of a subject having myofibrosis any composition comprising NRPC as described herein in an effective amount for treating the myofibrosis.

[0040] In further embodiments, the present disclosure provides a method for treating muscle inflammation, comprising administering to a subject having muscle inflammation an effective amount for treating the muscle inflammation, any composition comprising NRPC as described herein.

[0041] In further embodiments, the Disclosure provides a method for inducing angiogenesis in ischemic tissue, comprising administering to the body of a subject having ischemic tissue an effective amount for inducing angiogenesis of any composition comprising the NRPC described herein.

[0042] In further embodiments, the present disclosure provides a method for treating critical limb ischemia (CLI), comprising injecting into the body of a subject having CLI an effective amount for treating CLI, any composition comprising the NRPC described herein.

[0043] In further embodiments, the Disclosure provides a method for treating peripheral nerve injury, comprising administering into the body of a subject having peripheral nerve injury an effective amount for treating the peripheral nerve injury, any composition comprising the NRPC described herein.

[0044] In further embodiments, the Disclosure provides a method for suppressing the overexpression of peripheral myelin protein 22 (PMP22) in a subject. This method involves administering a composition comprising the NRPC described herein to a local area of ​​the body of a subject in which PMP22 overexpression is confirmed or evaluated, in an amount effective enough to suppress PMP22 overexpression in at least the local area.

[0045] In some embodiments of the present disclosure, which may be combined with any other embodiments, the method treats Charcot-Marie-Tooth disease (CMT) in question.

[0046] In some embodiments of this disclosure, which may be combined with any other embodiments, a composition comprising the NRPC described herein is administered into the body of a subject in an effective amount. In some embodiments, the composition being administered is in a frozen state. In other embodiments, the composition is administered in one or more passages after the NRPC has been thawed from its frozen state.

[0047] In further embodiments, the Disclosure provides a method for increasing miR-29a expression. This method involves administering a composition comprising the NRPC described herein to a local area of ​​the body in which the need for increased miR-29a expression is identified or evaluated, in an amount effective to increase miR-29a expression in at least the local area.

[0048] In one embodiment of the present disclosure, which may be combined with any other embodiment, increasing the expression of miR-29a results in suppression of the overexpression of peripheral myelin protein 22 (PMP22) at least in a local region.

[0049] In some embodiments of the present disclosure, which may be combined with any other embodiments, the method treats Charcot-Marie-Tooth disease (CMT) in question.

[0050] In this disclosure, the term “Subject” refers to, but is not limited to, individuals requiring administration of the compositions or nerve regeneration-promoting cells of this disclosure, including, mammals (e.g., humans), birds, reptiles, amphibians, fish, and the like.

[0051] In this disclosure, “treatment, procedure” means any action that improves or preferably alters the symptoms of a disease by administering the compositions relating to this disclosure.

[0052] Further features and advantages of the disclosed methods and apparatus will be described below, but these will be evident from the detailed description and drawings. The features and advantages described herein are not exhaustive, and many additional features and advantages will be evident to those skilled in the art in consideration of the figures and descriptions. Furthermore, it should be noted that the language used herein has been selected primarily for readability and explanatory purposes and does not limit the scope of the subject matter of the invention. [Brief explanation of the drawing]

[0053] [Figure 1]The exemplary embodiments of this disclosure show the population doubling times and cell sizes of different mesenchymal stem cells (MSCs) originating from various regions. [Figure 2] The cell size, viability, and doubling time of tonsil-derived mesenchymal stem cells (TMSCs) obtained from different humans according to exemplary embodiments of this disclosure are shown. [Figure 3] The marker expression rates of mesenchymal stem cells (MSCs) derived from different regions of human beings, and neuronal regeneration-promoting cells (NRPCs) differentiated from MSCs, according to exemplary embodiments of this disclosure. [Figure 4A] The cytokine expression rates between MSCs and NRPCs derived from different human regions are shown in exemplary embodiments of this disclosure. [Figure 4B] The cytokine expression rates between MSCs and NRPCs derived from different human regions are shown in exemplary embodiments of this disclosure. [Figure 5] A series of immunofluorescence images showing increased expression of nerve health-related proteins in NRPC obtained from different stages of the NRPC production process according to exemplary embodiments of the present disclosure. [Figure 6A] Exemplary embodiments of this disclosure demonstrate different levels of neurite outgrowth between MSCs and NRPCs originating from different regions of human beings. [Figure 6B] Exemplary embodiments of this disclosure demonstrate different levels of neurite outgrowth between MSCs and NRPCs originating from different regions of human beings. [Figure 7] These are a series of images showing different neurite outgrowth between tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs) according to exemplary embodiments of the present disclosure. [Figure 8A] A series of graphs are shown illustrating the average number of neurites and the average length of neurites in neurite growth assays performed between tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs) according to exemplary embodiments of the present disclosure. [Figure 8B]A series of graphs are shown illustrating the average number of neurites and the average length of neurites in neurite growth assays performed between tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs) according to exemplary embodiments of the present disclosure. [Figure 9A] The following are a series of graphs and images illustrating the reduction in neurite growth between T-MSC and T-NRPC samples when CD121a expression is reduced using small interfering RNA (siRNA) according to exemplary embodiments of the present disclosure. [Figure 9B] The following are a series of graphs and images illustrating the reduction in neurite growth between T-MSC and T-NRPC samples when CD121a expression is reduced using small interfering RNA (siRNA) according to exemplary embodiments of the present disclosure. [Figure 10A] The expression levels of the marker CD121a between working cell banks of MSCs and NRPCs originating from various regions, and between NRPCs themselves, according to exemplary embodiments of this disclosure. [Figure 10B] The expression levels of the marker CD121a between working cell banks of MSCs and NRPCs originating from various regions, and between NRPCs themselves, according to exemplary embodiments of this disclosure. [Figure 11A] The expression levels of the marker CD121a between T-MSCs and NRPCs across various passages are shown in exemplary embodiments of this disclosure. [Figure 11B] The expression levels of the marker CD121a between T-MSCs and NRPCs across various passages are shown in exemplary embodiments of this disclosure. [Figure 12] Figure 12 is a series of images and graphs illustrating the relationship between neurite outgrowth assays and marker CD121a expression levels between T-MSCs and NRPCs across various passages, according to exemplary embodiments of the present disclosure. [Figure 13A] A series of graphs showing the average number and average length of neurites of T-MSCs and NRPCs across various passages, according to exemplary embodiments of the present disclosure, are shown. [Figure 13B]A series of graphs showing the average number and average length of neurites of T-MSCs and NRPCs across various passages, according to exemplary embodiments of the present disclosure, are shown. [Figure 14A] The following are a series of graphs showing the expression of CD121a in T-MSC2009R and NRPC2009R corresponding to passages 15-19, according to exemplary embodiments of the present disclosure. [Figure 14B] The following are a series of graphs showing the expression of CD121a in T-MSC2009R and NRPC2009R corresponding to passages 15-19, according to exemplary embodiments of the present disclosure. [Figure 14C] The following are a series of graphs showing the expression of CD121a in T-MSC2009R and NRPC2009R corresponding to passages 15-19, according to exemplary embodiments of the present disclosure. [Figure 15A] The present disclosure includes a series of images showing the differentiation of MSCs from various human regions into NRPCs, as well as a series of graphs showing the expression rates of the marker CD121a between NRPCs and their corresponding MSCs from various human regions, according to exemplary embodiments of this disclosure. [Figure 15B] The present disclosure includes a series of images showing the differentiation of MSCs from various human regions into NRPCs, as well as a series of graphs showing the expression rates of the marker CD121a between NRPCs and their corresponding MSCs from various human regions, according to exemplary embodiments of this disclosure. [Figure 16A] The expression levels of the marker CD121a between working cell banks of MSCs and NRPCs originating from various regions, and between NRPCs themselves, according to exemplary embodiments of this disclosure. [Figure 16B] The expression levels of the marker CD121a between working cell banks of MSCs and NRPCs originating from various regions, and between NRPCs themselves, according to exemplary embodiments of this disclosure. [Figure 17A] This disclosure illustrates, in exemplary embodiments thereof, blood flow analysis over a period of time in animal samples having severe lower limb ischemia (CLI) and undergoing various forms of treatment based on MSC and NRPC. [Figure 17B]This disclosure illustrates, in exemplary embodiments thereof, blood flow analysis over a period of time in animal samples having severe lower limb ischemia (CLI) and undergoing various forms of treatment based on MSC and NRPC. [Figure 17C] This disclosure illustrates, in exemplary embodiments thereof, blood flow analysis over a period of time in animal samples having severe lower limb ischemia (CLI) and undergoing various forms of treatment based on MSC and NRPC. [Figure 18A] The present disclosure shows a series of images illustrating myofibrosis, muscle inflammation, and capillary formation over a period of time in animal specimens subjected to various forms of treatment, according to exemplary embodiments of this disclosure. [Figure 18B] The present disclosure shows a series of images illustrating myofibrosis, muscle inflammation, and capillary formation over a period of time in animal specimens subjected to various forms of treatment, according to exemplary embodiments of this disclosure. [Figure 19A] The results of nerve conduction studies performed on mouse samples using various levels of T-NRPC according to exemplary embodiments of this disclosure are shown. [Figure 19B] The results of nerve conduction studies performed on mouse samples using various levels of T-NRPC according to exemplary embodiments of this disclosure are shown. [Figure 19C] The results of nerve conduction studies performed on mouse samples using various levels of T-NRPC according to exemplary embodiments of this disclosure are shown. [Figure 20A] The images of three sets of immunochemically stained sciatic nerves from mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure show the expression of various protein markers. [Figure 20B] The images of three sets of immunochemically stained sciatic nerves from mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure show the expression of various protein markers. [Figure 20C] The images of three sets of immunochemically stained sciatic nerves from mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure show the expression of various protein markers. [Figure 21A] The following are five sets of images showing the G ratio and myelination of neurons in mouse samples treated with various levels of T-NRPC according to exemplary embodiments of the present disclosure. [Figure 21B] The following are five sets of images showing the G ratio and myelination of neurons in mouse samples treated with various levels of T-NRPC according to exemplary embodiments of the present disclosure. [Figure 21C] The following are five sets of images showing the G ratio and myelination of neurons in mouse samples treated with various levels of T-NRPC according to exemplary embodiments of the present disclosure. [Figure 21D] The following are five sets of images showing the G ratio and myelination of neurons in mouse samples treated with various levels of T-NRPC according to exemplary embodiments of the present disclosure. [Figure 21E] The following are five sets of images showing the G ratio and myelination of neurons in mouse samples treated with various levels of T-NRPC according to exemplary embodiments of the present disclosure. [Figure 22A] Three sets of images and graphs are shown illustrating the expression of markers PMP22 and MPZ among mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure. [Figure 22B] Three sets of images and graphs are shown illustrating the expression of markers PMP22 and MPZ among mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure. [Figure 22C] Three sets of images and graphs are shown illustrating the expression of markers PMP22 and MPZ among mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure. [Figure 22D] Three sets of images and graphs are shown illustrating the expression of markers PMP22 and MPZ among mouse samples treated with various levels of T-NRPC according to exemplary embodiments of this disclosure. [Figure 23A]A table illustrating the analysis of several miRNAs that appeared to be expressed in TMSC and T-NRPC cultures according to exemplary embodiments of this disclosure is shown. [Figure 23B] A table illustrating the analysis of several miRNAs that appeared to be expressed in TMSC and T-NRPC cultures according to exemplary embodiments of this disclosure is shown. [Modes for carrying out the invention]

[0054] The subject matter disclosed herein is described and discussed in more detail with reference to several specific embodiments and examples, which include some, but not all, embodiments of the invention. Similar figures refer to similar components or parts throughout. The subject matter disclosed herein may be embodied in many different forms and should not be construed as being limited to the specific embodiments described herein. Rather, these embodiments are provided to satisfy the legal requirements to which this disclosure is applicable. Indeed, many modifications and other embodiments of the subject matter disclosed herein will come to mind for those skilled in the art to which the subject matter of this disclosure relates. Therefore, it should be understood that the subject matter disclosed herein should not be limited to the specific forms disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0055] Various neurological disorders arise from inadequate or insufficient myelin formation. Myelin is a lipid-rich material that surrounds the axons of nerve cells (for example, as a myelin sheath), insulating them and increasing the rate at which action potentials propagate along the axons. Myelin is formed by oligodendrocytes and glial cells such as Schwann cells. Various neurological disorders can result from inadequate or insufficient myelination of nerve cells, or insufficient growth of axons and other neurites. For example, Charcot-Marie-Tooth (CMT) disease is a genetic disorder that occurs in 1 in 2,500 people, and its phenotype and genetic causes are heterogeneous. CMT1A type (CMT1A) is a type of hereditary neurological disorder that affects peripheral nerves and is caused by duplication of the peripheral myelin protein 22 (PMP22) gene.

[0056] Such neurological disorders highlight the need for more effective stem cell treatments. Damaged nerves, whether due to inadequate or insufficient myelin formation or insufficient neurite growth, can be replaced or regenerated using stem cells. Specifically, mesenchymal stem cells (MSCs) are widely used in the development of cell therapies because they can differentiate into various cell types in response to specific stimuli. The cell types associated with the recovery of damaged nerves may require the effective expression of proteins involved in nerve replacement, regeneration, and / or myelin formation. However, the ability of MSCs to effectively differentiate into suitable precursor cells (referred herein to as “neuroregenerating proliferative cells (NRPCs)”) and subsequently express proteins responsible for nerve replacement, regeneration, and / or myelin formation is challenging due to various factors. For example, such proteins expressed by NRPCs derived from differentiated MSCs are often expressed in low yields, or neurite growth resulting from NRPC samples may be insufficient.

[0057] This disclosure describes a novel and improved NRPC for treating damaged neurons, and a method for generating it. This disclosure describes more effective neuronal receptacles (NRPCs) for treating damaged neurons and methods for generating such NRPCs. The inventors of this disclosure have found that NRPCs differentiated from MSCs obtained from specific human regions (e.g., tonsils) appear to have favorable expression rates of relevant proteins for treating nerves damaged due to the aforementioned neurological disorders. Such MSCs may be referred to herein as tonsil-derived MSCs (T-MSCs). Furthermore, the inventors have found that NRPCs derived from T-MSCs expressing the protein markers CD26, CD106, CD112, CD121a, and CD141 (referred to herein as T-NRPCs) have a higher success rate in treating damaged nerves, and methods for selectively growing such T-NRPCs expressing such proteins are disclosed. In particular, the inventors have found that selectively growing T-NRPCs expressing the protein marker CD121a above a threshold level (e.g., above 30% at the working cell bank (WCB) stage) significantly increases the effectiveness of NRPCs for treating damaged nerves. Furthermore, the inventors discovered that selectively screening T-NRPC samples for those exhibiting neurite formation and promoting their proliferation leads to a more effective product for treating damaged nerves.

[0058] The origin of MSC is related to the effective production of NRPC. As discussed herein in conjunction with the experimental data described herein, the inventors have found that the origin of MSCs is related to the generation of effective NRPCs. For example, the region of the human body from which MSCs are collected has an effect on the ability of MSCs to effectively differentiate into NRPCs and / or to effectively express related protein markers.

[0059] MSCs from various regions of human organisms may express the same relevant protein markers, but the doubling times of the population tend to differ among MSCs. For example, Figure 1 shows the population doubling times and cell sizes of different samples of mesenchymal stem cells (MSCs) derived from various regions of the human body according to exemplary embodiments of the present disclosure. These various regions include the amygdala, adipose tissue, bone marrow, and umbilical cord, from which amygdala-derived MSCs (T-MSCs), adipose-derived MSCs (AD-MSCs), bone marrow-derived MSCs (BM-MSCs), and umbilical cord-derived MSCs (UC-MSCs), respectively. Effectively generating NRPCs from MSCs depends on the ability of the MSCs to proliferate to population size in order to allow a sufficient number of MSCs to differentiate into the desired NRPC candidate cells. The population size proliferation of any cell sample can be measured by determining the population doubling time of the cell sample, which is the time (e.g., several hours) it takes for the number of cells in the cell sample to double. Alternatively, proliferation can be measured by determining the population doubling level (PDL) of the cell sample, which is the total number of times the cells in a given population have doubled during in vitro culture. As shown in Figure 1, proliferation appears to differ among different MSC samples. For example, based on population doubling time and population doubling level, UC-MSCs and T-MSCs were found to have the highest population size growth. However, regardless of proliferation rate, the expression levels of characteristic markers indicating that cells are MSCs (the markers may be CD73, CD90, and CD105) were found to be nearly identical across each of the four MSC samples. These expression levels confirmed the presence of MSCs in samples taken from four human regions (tonsils, adipose tissue, bone marrow, and umbilical cord). While the expression levels of such proteins confirmed that the obtained cell samples actually contained MSCs, the expression levels of other proteins indicate that T-MSCs produce more effective NRPCs compared to other MSCs, as will be discussed herein.

[0060] Cell size, cell viability, and population doubling time were measured for MSCs obtained from different human amygdala regions. As already discussed, the origin of MSCs is relevant to producing effective NRPCs because the specific human region from which MSCs are obtained (e.g., tonsils, adipose tissue, bone marrow, umbilical cord, etc.) can provide clues to the effectiveness of NRPCs and / or the MSCS used to generate NRPCs. Specifically, as described in the following studies, we found that tonsil-derived MSCs are most effective in generating NRPCs (T-NRPCs) and yield the best results for nerve regeneration (e.g., measured by neurite outgrowth). To begin the study, we obtained MSCs from the same region of different humans (e.g., tonsils). However, we tested the obtained MSCs to confirm that they were consistent with respect to cell size, cell viability, and population doubling time. We anticipated that differences in cell size, cell viability, and population doubling time could affect the effectiveness of the study (e.g., by introducing unintended variables), and therefore we evaluated the consistency within the samples of obtained T-MSCs. Figure 2 shows the cell size, viability, and doubling time of tonsil-derived mesenchymal stem cells (T-MSCs) obtained from different humans. The T-MSCs were identified as 2001L, 2001R, 2005R, 2009L, and 2009R, each identification indicating the human (e.g., numbered 2001, 2005, 2009, etc.) and the left or right tonsil (e.g., L or R) from which the T-MSC originated. Specifically, population doubling levels, cell size, and cell viability were measured across passages for T-MSC samples obtained from humans identified as at least 2001L, 2005R, 2009L, and 2009R. Population doubling levels appeared to increase with increasing passage number in all T-MSC samples, except in the human 2009L-derived T-MSC 2009L sample, where the rate of increase was lower. Cell size, measured by cell diameter (in micrometers), appeared to remain consistent across T-MSC samples. Cell viability, measured by trypan blue staining, also appeared to remain consistent across all T-MSC samples. Nevertheless, there were no significant differences in cell size, viability, or population doubling time between any given passaged samples.Therefore, the inventors were able to ensure the consistency of T-MSC samples obtained from humans.

[0061] Use of subculturing in the processes described herein As used herein, passage may refer to the process by which a cell culture from a sample is passaged, i.e., taken and reseeded into one or more “daughter” cell culture flasks, the reseeded cells being deployed again into cell cultures in each daughter cell culture flask. This process may be repeated, and as a result, cell cultures from “daughter” cell culture flasks may be passaged again in subsequent “daughter” cell culture flasks (or other suitable containers). The passage number may indicate the number of these repetitions. For example, passage 5 (P5) indicates that cells from an existing cell culture have been taken and reseeded five times into new cell culture flasks (to be deployed again into new cell cultures). As described herein, a method for producing NRPCs disclosed for the treatment of damaged nerves may include culturing and passaged cell samples (e.g., MSCs, candidate NRPCs, and NRPC samples) over many passages. By doing so, each subsequent cell culture is replenished with fresh growth medium, thereby optimizing the growth, health, and stability of the cell cultures.

[0062] The expression rate of CD121a varies depending on the origin of the MSC. As previously discussed, the inventors have discovered that novel and improved NRPCs for treating damaged cells can be generated by selectively screening NRPCs that express specific marker proteins, particularly CD121a. The inventors have also found that the expression rate of CD121a varies depending on the origin of the MSCs that differentiate into NRPCs. Figure 3 shows the marker expression rates of MSCs derived from various regions of human tissue and NRPCs differentiated from MSCs, according to exemplary embodiments of the present disclosure. The expression rates are shown through heatmap 310, table 320, and histogram chart 330. Heatmap 310 shows the expression rates of various marker proteins (including CD121a) related to the treatment of damaged nerves in red intensity. Heatmap 310 confirms that the expression rates of marker proteins differ depending on the origin of the MSC sample, and that the marker expression rates also differ among differentiated NRPC samples. Table 320 lists the actual expression rates of various marker proteins associated with the treatment of damaged nerve cells in MSC and NRPC samples, and histogram 330 shows these (using bar heights), which vary based on the origin of the sample. In particular, Table 320 and histogram 330 show that the expression rate of CD121a is higher in T-MSCs than in MSCs from other tissues (AD-MSCs, BM-MSCs, and UC-MSCs). Furthermore, Table 320 and histogram 330 show that the expression rate of CD121a is much higher in T-NRPCs than in AD-NRPCs. The inventors found that by selectively screening, culturing, and growing MSCs and / or T-NRPCs based on selection criteria defined by the expression rate of relevant marker proteins, particularly CD121a, the resulting T-NRPC products were significantly more effective in treating damaged nerve cells. Based on the experimental results shown in Figure 3, CD121a is expressed in 91% of T-NRPCs but only in 17% of AD-NRPCs (an expression level of approximately 17% may be below the threshold for selection, and therefore such samples may be considered "non-selectable" as described below).CD121a expression rates also differed among MSC samples. CD121a expression rates were higher among T-MSCs than among other MSCs. Expression rates increased similarly as MSCs from each region differentiated into their respective NRPCs, but the rate of increase varied based on the MSC's origin. In the case of adipose tissue-derived AD-MSCs, the increase in expression rate ranged from 1% (among AD-MSCs) to 17% (among AD-NRPCs), which was significantly lower than the increase in tonsil-derived T-MSCs (an increase of 37% (among T-MSCs) to 91% (among T-NRPCs)). These results suggest that T-NRPCs can be identified and selected based on their high CD121a expression rates.

[0063] Selectable samples versus non-selectable samples, based on the expression rates of each protein marker, whether they meet or do not meet the expression rate threshold. As discussed herein, cell samples (e.g., NPRCs and / or NRPC candidates) may be selected (e.g., considered “selectable”) or not selected (e.g., considered “unselectable”) based on the expression rate of the cell sample with respect to the protein marker of interest. Depending on the protein marker, different thresholds may be set for the expression rate of that protein marker. A cell sample is considered selectable if the expression rate of a given protein marker by a given cell sample exceeds the threshold expression rate assigned to that protein marker. However, a cell sample is considered unselectable if the expression rate of a given protein marker by a given cell sample falls below the threshold expression rate assigned to that protein marker. Furthermore, the ability of cells to express a protein may depend on the state of the cell in which the protein expression level is measured.

[0064] For example, these cells may be in a frozen state, and the protein expression level in these frozen cells can be determined and / or estimated by measuring the protein expression level within these cells immediately after thawing. Measuring the expression level immediately after thawing avoids heat-dependent cellular activities (e.g., enzyme activity) that may affect the expression rate. The post-thaw period that can be considered "immediately after thawing" may be within approximately 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes after thawing. In embodiments, the post-thaw time forming the upper limit of the time considered to be immediately after thawing may be within a range formed by selecting any two numbers (2) listed in the preceding sentence (for example, the post-thaw time forming the upper limit of the time considered to be immediately after thawing may be about 1 minute to about 5 minutes, about 30 seconds to about 10 minutes, about 2 minutes to about 4 minutes, etc.). Furthermore, or alternatively, the “immediately after thawing” time of a cell may be before any passage after the cell has been thawed. In embodiments, the threshold at which a cell sample becomes selectable based on the expression rate or level at which a protein marker is expressed may be higher for unfrozen or “live” cells than for frozen cells (e.g., as a product). Unfrozen or “live” cells may include cells that have been passaged one or more times after being thawed from a frozen state. Other protein markers may have different (e.g., lower or higher) thresholds for expression rates to determine whether cells expressing such protein markers are selectable or not. In some embodiments, cells that are not frozen or are viable may not have been previously frozen, and therefore may not require any thawing process before any one or more passages before any measurement of protein expression rates.

[0065] Selectivity may depend on a first threshold for CD121a expression at an early stage of the NRPC production process. For example, in at least some embodiments, a cell sample may be selected (e.g., considered “selectable”) if it expresses CD121a at an expression rate above a first threshold (e.g., 30%) when the cells are frozen (e.g., during the working cell bank stage), but above a second threshold (e.g., 80%) when the cells are not frozen or viable (e.g., during the product stage). The different thresholds of protein expression required for the selection of a cell sample may depend on the stage in the NRPC production process the cell sample is in; therefore, the lower threshold required for protein expression may be lower if the cell sample is in an earlier stage, but a higher threshold may be required for protein expression if the cell sample is in a later stage. In particular, the lower threshold for CD121a expression may be set for the selection and proliferation of cell samples in the working cell bank stage. Cell samples selected in this earlier stage (referred to herein as “NRPC candidates”) may be frozen and may contain MSCs in the process of differentiation into their respective NRPCs. Furthermore, or alternatively, NRPC candidates may already be differentiated into NRPCs but may require further development and / or differentiation. For example, the inventors have found that NRPC candidates at the working cell bank stage, having a CD121a expression level higher than a first threshold rate of approximately 30%, can ultimately be differentiated into desired NRPC samples capable of expressing CD121a above a second threshold at a later stage (e.g., the product stage). However, in some embodiments, the first threshold for CD121a expression level at this early stage may or may not be higher. For example, an NRPC candidate may be selected if its CD121a expression level is at least above a threshold, i.e., approximately 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, 70%, 72.5%, 75%, 77.5%, or 80%.In the embodiment, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC candidate sample may be selected if the CD121a expression rate is at least above a threshold such as about 30%, about 30% to about 40%, or about 35% to about 50%).

[0066] Selectivity may also depend on a second threshold for CD121a expression at an early stage of the NRPC production process. Using a second threshold for CD121a expression, selection can be made from NRPC products, which may be living or unfrozen, as previously discussed. For example, we have found that NRPC candidates that have undergone one or more passages after thawing at the product stage and have a CD121a expression level higher than the second threshold rate of about 80% were able to ultimately form improved NRPCs that provide the advantages described herein. However, in some embodiments, the second threshold for CD121a expression level at this later stage may be higher or lower. For example, with respect to NRPCs at a later stage (e.g., the product stage and / or "living" state), an NRPC may be selectable if it expresses CD121a at an expression rate at least above the second threshold, i.e., about 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In the embodiment, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, cells may be selected if the expression rate of CD121a is at least above a threshold of about 70%, about 72.5% to about 77.5%, about 85% to about 95%, etc.).

[0067] Some MSCs and NRPCs could not be selected because they did not meet the CD121a expression threshold. Figure 3 shows the expression rates of various protein markers, including CD121a, in MSCs and NRPCs. As shown in Figure 3, CD121a expression increased in some cell samples as MSCs differentiated into NRPCs. For example, CD121a expression in adipose tissue-derived NRPCs (AD-NRPCs) was higher than that of adipose-derived MSCs (AD-MSCs) from which AD-NRPCs differentiated. However, despite the increase, CD121a expression was not substantial enough to consider AD-MSCs or AD-NRPCs as selectable. As shown in Table 320 and histogram 330 in Figure 3, AD-MSC, bone marrow-derived MSCs (BM-MSCs), and umbilical cord-derived MSCs (UC-MSCs) also failed to express the CD121a protein marker at sufficiently high expression rates (e.g., over approximately 30%) at earlier stages, and were therefore not selectable. In contrast, CD121a expression was higher in tonsil-derived MSCs (T-MSCs) than in MSCs from other tissues. The inventors found that T-MSC samples could meet a first threshold (e.g., about 30%) by expressing CD121a at 37.04%, and were therefore considered selectable. The inventors also found that the CD121a expression rate increased as T-MSCs differentiated into their respective NRPCs (T-NRPCs). Live T-NRPC samples obtained at the product stage (e.g., after the first subculturing after thawing (e.g., subculturing)) after selectively differentiating T-MSC samples into their respective T-NRPCs could also successfully express the CD121a protein marker by expressing CD121a at 91.26%, exceeding a second threshold (e.g., about 80%), and were therefore also considered selectable.

[0068] The expression levels of other proteins (CD26, CD106, CD112, and CD141) were also correlated. While CD121a was observed to be significantly more highly expressed by T-NRPCs, Figure 3 also shows other relevant trends in protein expression rates when various MSCs differentiate into their respective NRPCs. For example, when comparing the protein marker expression patterns of MSCs with those of the respective NRPCs from which the MSCs differentiated, certain CD markers are associated with a significant increase or decrease in expression rates when MSCs differentiate into their respective NRPCs. As shown in Figure 3, CD markers that showed increased expression in NRPCs (compared to the respective MSCs from which the NRPCs originated) included CD106 and CD112 (in addition to CD121a). This increase in expression was most pronounced in tonsil-derived NRPCs (T-NRPCs) differentiated from tonsil-derived MSCs (T-MSCs). Furthermore, as shown in Figure 3, CD markers that showed decreased expression in NRPCs (compared to the respective MSCs from which the NRPCs originated) included CD26 and CD141. Therefore, patterns of increased (e.g., CD106, CD112, and CD121a) or decreased (CD26 and CD141) expression of these CD markers are useful for identifying NRPCs differentiated from MSCs. While the generally altered expression of markers CD121a, CD106, CD112, CD26, and CD141 can be used as differentiation markers for pro-neuroregenerative cells, the significantly high expression rate of CD121a is particularly useful for identifying effective NRPCs, as discussed herein.

[0069] NRPCs may be selected based on the expression levels of CD106, CD112, and CD121a when an NRPC or NRPC candidate is in the product stage. NRPCs differentiated from MSCs can be selected by identifying increased expression rates of CD106, CD112, and CD121a, and decreased expression rates of CD26 and CD141. As shown in a specific example in Figure 3, T-NRPCs expressed CD106 at 20.13%, CD112 at 59.93%, and CD121a at 91.26%, based on measured expression levels of the aforementioned proteins when the T-NRPCs were in a living, unfrozen state at the product stage (e.g., after one passage after thawing). In some embodiments, NRPCs may be selected based on the CD106 expression rate being at least above a threshold of approximately 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, or 40%. In embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC may be selected if the CD106 expression rate is at least greater than about 10%, about 10% to about 30%, about 15% to about 25%, etc.). In some embodiments, an NRPC may be selected based on a CD112 expression rate at least above a threshold of about 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%. In embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC may be selected if the CD121a expression rate is about 25%, about 25% to about 40%, about 30% to about 50%, etc.). As already discussed, effective T-NRPCs may be identified and selected based on high expression rates in CD121a expression. For example, T-NRPCs may be identified and selected based on the CD121a expression rate, which exceeds at least a certain threshold while the T-NRPC is in an unfrozen state (e.g., one or more passages after thawing). This threshold may be approximately 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%.In embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, a T-NRPC may be identified and selected if CD121a is expressed at least above a threshold of about 80%, about 82.5% to about 87.5%, about 85% to about 95%, etc.).

[0070] NRPCs may be selected based on the expression levels of CD106, CD112, and CD121a when NRPCs or NRPC candidates are in their working cell bank stage. In some embodiments, T-NRPCs can be selected based on their expression rate when T-NRPCs or T-NRPC candidates are in the working cell bank stage (e.g., immediately after thawing from a frozen state (e.g., before any subsequent passage after thawing)). As already discussed, the expression rate of a protein in a frozen cell can be determined and / or estimated by measuring the expression rate of the protein in that cell immediately after thawing from the frozen state. Measuring the expression rate immediately after thawing can avoid heat-dependent cellular activities (e.g., enzyme activity) that may affect the expression rate. The post-thaw period that can be considered "immediately after thawing" may be within approximately 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes after thawing. In embodiments, the post-thaw time forming the upper limit of the time considered to be immediately after thawing may be within a range formed by selecting any two numbers (twice) listed in the preceding sentence (for example, the post-thaw time forming the upper limit of the time considered to be immediately after thawing may be about 1 minute to about 5 minutes, about 30 seconds to about 10 minutes, about 2 minutes to about 4 minutes, etc.). For example, T-NRPCs may be identified and selected based on the fact that the CD106 expression rate is at least above a threshold, which may be about 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, or 35%, and the CD106 expression rate is measured when the T-NRPC or T-NRPC candidate is in the working cell bank stage. T-NRPCs may be identified and selected based on the CD112 expression rate being at least above a threshold, which may be approximately 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, or 70%, where the CD112 expression rate is measured when the T-NRPC or T-NRPC candidate is in the working cell bank stage. As already discussed, T-NRPCs or T-NRPC candidates may also be selected based on the threshold expression level of CD121a when the T-NRPC or T-NRPC candidate is in the working cell bank stage.For example, a T-NRPC or T-NRPC candidate may be selected if its CD121a expression level is at least above a threshold, i.e., approximately 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, 70%, 72.5%, 75%, 77.5%, or 80%. In embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC candidate sample may be selected if the CD121a expression rate is at least above thresholds such as approximately 30%, approximately 30% to approximately 40%, or approximately 35% to approximately 50%).

[0071] NRPCs may be selected based on the expression levels of CD26 and CD141 when an NRPC or NRPC candidate is in the product stage. T-NRPCs can be selected based on measuring the expression levels of CD26 and CD141 when the T-NRPC is in the product stage (e.g., one or more passages in a live, unfrozen state after thawing). For example, in the specific experiment shown in Figure 3, T-NRPCs are selected based on expression rates of 21.77% for CD26 and 23.75% for CD141.

[0072] In some embodiments, T-NRPCs may be identified and selected based on the CD26 expression rate being at least above a certain threshold, which may be approximately 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35%. In embodiments, the threshold may be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC may be selected if the CD26 expression rate is approximately 10%, approximately 10% to approximately 20%, approximately 15% to approximately 25%, etc.).

[0073] In some embodiments, T-NRPCs may be identified and selected based on the fact that the CD141 expression rate is at least above a certain threshold, which may be about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In the embodiment, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, NRPC may be selected if the CD141 expression rate is about 10%, about 10% to about 20%, about 15% to about 25%, etc.).

[0074] NRPCs may be selected based on CD26 and CD141 expression levels when NRPCs or NRPC candidates are in their working cell bank stage. In some embodiments, T-NRPCs can be selected based on their expression rate when a T-NRPC or T-NRPC candidate is in the working cell bank (WCB) stage (e.g., immediately after thawing from a frozen state (e.g., before any subsequent passage after thawing)). At the WCB stage, T-NRPCs may be identified and selected if the CD26 expression rate is less than 5%. T-NRPCs may be identified and selected based on the CD26 expression rate being at least below a certain threshold, which may be approximately 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 4.75%, 4.8%, 4.85%, 4.9%, 4.95%, or 5%. At the WCB stage, T-NRPCs may be identified and selected if the CD141 expression rate is less than 30%. T-NRPCs may be identified and selected based on the CD141 expression rate being at least below a certain threshold, which may be approximately 1%, 5%, 10%, 15%, 20%, 25%, 28%, 28.5%, 29%, 29.5%, or 30%. In the embodiment, T-NRPC may be identified and selected when the expression rate of CD26 is less than 5% and the expression rate of CD141 is less than 30%.

[0075] In embodiments, T-NRPCs may be selected based on thresholds different from those in the embodiments described above. In certain embodiments, T-NRPCs may be identified and selected based on the CD26 expression rate being at least below a certain threshold, which may be about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, or 35%, and the CD26 expression rate is measured when the T-NRPC or T-NRPC candidate is in the working cell bank stage. In embodiments, T-NRPCs may be identified and selected based on the CD141 expression rate being at least below a certain threshold, which may be about 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, or 50%, and the CD141 expression rate is measured when the T-NRPC or T-NRPC candidate is in the working cell bank stage.

[0076] NRPCs can be selected based on characteristic expression patterns of CD26 and CD141. Alternatively, T-NRPCs can be identified based on the difference in CD26 and CD141 expression rates by the NRPC compared to those by the respective MSCs. That is, a characteristic expression pattern by a desired NRPC (e.g., T-NRPC) may show a decrease in expression rate compared to the respective MSCs from which the NRPC was differentiated. For example, in a specific example shown in Figure 3, the CD26 expression rate was reduced from 48.48% by the T-MSC to 21.77% by the T-NRPC (a reduction of 26.71%), and the CD141 expression rate was reduced from 60.36% by the T-MSC to 23.75% by the T-NRPC (a reduction of 36.61%). In contrast, CD26 expression was reduced from 90.03% by AD-MSCs to 27.98% by AD-NRPCs (a reduction of 62.05%), and CD141 expression actually increased from 36.95% by AD-MSCs to 44.96% by AD-NRPCs (an increase of 8.01%). In some embodiments, T-NRPCs may be identified and selected based on the reduction in CD26 expression (i.e., delta) between the CD26 expression rate by the NRPC and the CD26 expression rate by the respective MSCs. The reduction in CD26 expression may exceed at least a certain threshold, which may be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC may be selected if the reduction in CD26 expression between the CD26 expression rate by the NRPC and the CD26 expression rate by each MSC is at least about 10%, about 10% to about 20%, about 15% to about 25%, etc.). In some embodiments, T-NRPCs may be identified and selected based on the reduction in CD141 expression rate (i.e., delta) between the CD141 expression rate by the NRPC and the CD141 expression rate by each MSC. The reduction in CD141 expression rate may exceed at least a certain threshold, which may be about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%.In embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, NRPC may be selected if the reduction in CD141 expression between the CD26 expression rate by NRPC and the CD141 expression rate by each MSC is at least about 20%, about 20% to about 30%, about 25% to about 45%, etc.).

[0077] T-NRPC was found to express cytokines related to nerve regeneration at significantly higher levels than other NRPCs. Cytokines are small molecules of protein that are important for cellular signaling. In particular, it is well known that various cytokines promote signaling between NRPCs (neurophosphorus-reactive cells) and other cells for the treatment of damaged nerve cells. Examples of such cytokines include hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), and growth-regulating oncogene alpha (GRO-α). To provide NRPCs that are more effective in treating damaged nerve cells, it is useful to screen NRPCs for their ability to express such cytokines at sufficient expression rates. The inventors tested MSCs (method-derived MSCs from the amygdala and AD-MSCs from adipose tissue) derived from various regions of human cells, as well as their respective NRPCs (T-MSCs and AD-MSCs), for their ability to adequately express such cytokines (HGF, uPA, and GRO-α). Expression rates were compared with a control group (shown as "primary Schwann cells" in heatmap 410 of Figure 4A). Figures 4A and 4B show cytokine expression rates among MSCs and NRPCs derived from various regions via heatmap 410 and graphs 420A-C according to exemplary embodiments of the present disclosure. We found that T-NRPCs expressed cytokines related to nerve regeneration at significantly higher levels than other NRPCs differentiated from MSCs derived from other regions of human tissue. In both adipose tissue-derived and tonsil-derived sample groups (e.g., MSCs and NRPCs), NRPC samples expressed the aforementioned cytokines more highly than each of the MSC samples from which the NRPC samples were differentiated. For example, T-NRPC samples were the only samples among the others (AD-MSC, AD-NRPC, and T-MSC samples) that expressed HGF cytokines. AD-NRPC samples expressed uPA at a higher rate (i.e., 300%) than AD-MSC samples, but T-NRPC samples expressed uPA at a much higher rate (i.e., 1400%) than any of the other samples, including T-MSC samples.Furthermore, AD-MSC samples expressed GRO-α (i.e., at an intensity of 5000%), and while T-MSC samples showed only slight expression of GRO-α, T-NPCs expressed GRO-α at a higher rate than either sample, at 7000%. Therefore, based on the expression rates of cytokines related to nerve regeneration, we found that T-NRPCs are a superior NRPC option compared to AD-NRPCs for treating damaged nerve cells.

[0078] NRPC can be used to improve myelin formation and nerve stability by co-culturing NRPC with dorsal root ganglion (DRG) neurons. Dorsal root ganglion (DRG) neurons are clusters of neurons located at the dorsal roots of spinal nerves. It has been found that when NRPCs (neuroplastic polyposis cells) are co-cultured with DRG neurons, NRPCs can be induced to transform into Schwann cell-like cells capable of myelinating (e.g., covering) the axons of DRG neurons. As previously discussed, myelination around the axon insulates the axon, allowing electrical signals to propagate more efficiently over long distances through the neuron. Therefore, by promoting myelination in DRG neurons, NRPCs can treat damaged nerve cells by myelinating damaged neurons and / or by forming new myelinating neurons.

[0079] T-NRPC can be evaluated for its ability to induce myelination based on MBP expression in the dorsal root ganglia. Based on these experiments, it was discovered that the ability of NRPC candidates to induce myelination can be evaluated by detecting the expression of key proteins such as MBP and / or precursors such as NF-H. For example, NRPCs and / or NRPC candidates can be co-cultured with dorsal root ganglion cells. The ability of NRPCs and / or NRPC candidates to induce myelination can be evaluated by subsequently examining the dorsal root ganglia and confirming myelination based on the expression of MBP protein in the sample containing the dorsal root ganglia. In some embodiments, MBP protein expression can be detected by immunofluorescence, for example, after performing immunofluorescence staining and checking for immunofluorescence sensitivity. Since the generation, development, and myelination of nerve fibers and other supporting cells from the dorsal root ganglia are influenced by cell culture conditions, co-culture of dorsal root ganglia and NRPC candidate samples can be used to evaluate the ability of each NRPC candidate sample to myelinate the supporting cells of the dorsal root ganglia. Although dorsal root ganglia were used in this experiment, it is known that dorsal root ganglia induce the generation, development, and myelination of nerve fibers and other supporting cells, at least in humans. Therefore, it is intended that the ability of NRPC candidate samples to induce myelination in other species can be evaluated using cells equivalent to those found in dorsal root ganglia in other species.

[0080] When NRPCs are co-cultured with DRG neurons, they can more effectively promote myelin formation as the process progresses through the NRPC production stage. NRPC can differentiate and myelinate the axons of DRG neurons, or improve the stability of DRG neurons through the expression of related proteins such as MBP and TuJ1. Specifically, MBP is important in the nerve myelination process, and TuJ1 contributes to the stability of nerve cell bodies and microtubules. However, the inventors discovered that the expression rate of such proteins is increased, and that NRPC is in the production process. Specifically, the inventors obtained NRPC samples from three different stages of the NRPC production process (i.e., from the master cell bank (MCB), working cell bank (WCB), and product stage), co-cultured the obtained samples with DRG neurons, and observed the expression of proteins associated with myelination and improved neuronal stability. The inventors also obtained MSC samples (from passage 14) to serve as a control. The rate of protein expression was indicated by the fluorescence intensity measured via a fluorescent marker on the NRPC sample. Figure 5 is a series of immunofluorescence images showing increased expression of nerve regeneration-related proteins in NRPC obtained from three stages of the NRPC production process according to exemplary embodiments of this disclosure. As shown in Figure 5, the stages of the NRPC production process from which NRPC samples were obtained are passage 14 of a T-MSC culture (T-MSC P14) to serve as a control, a T-NRPC sample obtained from a master cell bank (MCB), a T-NRPC sample obtained from a working cell bank (WCB), and the product. As used herein, the master cell bank of T-NRPC contains aliquots of a single pool of T-NRPC cells differentiated from T-MSCs under specified conditions, dispensed into multiple containers, and stored under specified conditions. The working cell bank (WCB) of T-NRPC is obtained using the MCB. Thus, T-NRPC derived from the WCB is in a later stage of the production process compared to T-NRPC from the MCB. This product contains T-NRPC that can be readily used for treating damaged cells and is derived from WCB, thus producing T-NRPC from the product at a later stage of the production process compared to WCB-derived T-NRPC.After co-culturing T-NRPC samples derived from each stage with DRG neurons and measuring the expression of various proteins via immunofluorescence, the inventors discovered that MBP and TuJ1 expression increased throughout each stage of the T-NRPC production process (i.e., expression rates increased from T-MSC P14 to MCB, from MCB to WCB, and from WCB to product). The "Nuclei" column in Figure 5 shows the results of nuclear staining of DRG neurons co-cultivated with T-NRPC samples derived from each stage of the NRPC production process. As shown from the stained nuclei in the "Nuclei" column, the number of nuclei that are indicator DRG neurons changes at each stage of the NRPC production process. The "Merge" column in Figure 5 visualizes an integrated image of MBP and TuJ1 protein expression in addition to nuclear localization. By analyzing the information from the "Nuclei" and "Merge" columns, insights into the movement of cells involved in myelin formation can be obtained. Increased expression of MBP and TuJ proteins enabled enhanced cell communication via myelination and induction of neural tube formation.

[0081] The effectiveness of NRPC in inducing neurite growth can be determined using a neurite extension assay that includes neuroblastoma cells. Neurite processes (or neuronal processes) are known to protrude from the cell body of a neuron and are involved in the transport of axons, neurotransmitters, nerve growth factors, and other substances necessary for growth and regeneration. Neurite extension assays can be performed to compare neurite growth induced by nerve regeneration-promoting cells such as those described in this disclosure. Certain clonal lines of mouse neuroblastoma cells, such as N1E-115, are known to elongate or retract axons depending on the culture medium and may therefore be used in neurite extension assays to test the ability of the NRPCs of this disclosure to induce neurite growth.

[0082] Neurite extension assays demonstrated that T-NRPC was more successful in inducing neurite growth than other NRPCs. To test the effectiveness of various NRPCs in inducing neurite growth, N1E-115 cells (mouse neuroblastoma cells, ATCC, USA) were cultured and seeded on a microporous filter (neurite extension assay kit, Millipore, USA). The seeded cells were cultured in culture medium for 48 hours, and NRPC and MSC samples derived from various human regions were collected. The culture medium for each NRPC or MSC sample reflected the active components (e.g., proteins) expressed and / or produced by the respective NRPC or MSC sample. After staining the neurites that protruded through the microporous filter, the absorbance was measured. The neurite extension assay confirmed that NRPC cultures regulated or stimulated neurite (axon) growth in N1E-115 (mouse neuroblastoma) cells. However, we discovered that different NRPC and MSC samples differentially stimulated neurite growth. Figures 6A and 6B show a series of images of the obtained cultures 610 and graphs 620 and 630 that quantify neurite growth, illustrating these different levels of neurite elongation in MSCs and NRPCs from various regions. The different samples for which results are shown are AD-MSC, T-MSC, AD-NRPC, T-NRPC, negative control group, positive control group, and primary Schwann cell group. Graph 620 shows the average number of neurites between cells in each sample group, while graph 630 shows the length of the longest neurite (μm) in each sample group. We found that in the neurite elongation assay group using culture medium for T-NRPC samples, the number of axons and the length of the longest axon were significantly increased compared to the remaining groups in the neurite elongation assay group using culture medium for the remaining samples (T-MSC, AD-MSC, and AD-NRPC samples). Therefore, it was confirmed that neuronal development can be significantly enhanced by T-NRPC, as demonstrated by neurite outgrowth on N1E-115 neuroblastoma cells induced in the culture medium of T-NRPC samples.

[0083] There is variation in the ability to induce neurite outgrowth between NRPC and MSC samples derived from the tonsils. Neurite extension assays were also performed using culture media from different samples of tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs). Different samples corresponded to MSCs obtained from different individuals (indicated in the sample name by human identification information (e.g., given by numbers such as 2001, 2005, etc.)) and / or different sides of the tonsil (indicated in the sample name with "L" for the left tonsil and "R" for the right tonsil). To test the effectiveness of the culture media for these various T-NRPC and T-MSC samples in inducing neurite growth, N1E-115 cells (mouse neuroblastoma cells, ATCC, USA) were cultured and seeded on a microporous filter (neurite extension assay kit, Millipore, USA). The seeded cells were cultured in culture medium for 48 hours, from which various T-NRPC and T-MSC samples were induced from different individuals or sides of the tonsil. After staining the protruding neurites through a microporous filter, the absorbance was measured. The results of this experiment are shown in Figures 7, 8A, and 8B. Specifically, Figure 7 is a series of images showing different neurite elongation between tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs). Figures 8A and 8B present a series of graphs that quantify neurite elongation based on the average number of neurites and the average length of the longest neurite in a group of neurite growth assays corresponding to the culture media of different tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs). Furthermore, each T-NRPC sample may correspond to the respective T-MSC sample from which it originates, and each pair of samples (T-NRPC sample and its corresponding T-MSC sample) corresponds to an individual human (identifiable by numbers such as 2001, 2005, 2009, etc.) and the tonsil side (identifiable by "L" for left and "R" for right in the sample name). The results shown in Figures 7, 8A, and 8B confirm that, as the T-MSC samples differentiated into their respective T-NRPC samples, there was generally an increase in neurite outgrowth in N1E 115 cells induced by the culture medium of the sample.Therefore, the culture medium for T-NRPC2001L induced more neurite elongation than the culture medium for T-MSC2001L; the culture medium for T-NRPC2001R induced greater neurite elongation than the medium for T-MSC2001R; the culture medium for T-NRPC2009L induced greater neurite elongation than the culture medium for T-MSC2009L; and the culture medium for T-NRPC2009R induced greater neurite elongation than the culture medium for T-MSC2009R. There was an increase in neurite elongation, which can be measured by the increase in the average number of neurites in the induced N1E115 cell samples or by the length of the longest neurite in the induced N1E115 cell samples. However, there were outliers for T-MSC2005R and T-NRPC2005R. Based on the results of the neurite outgrowth assay shown in Figures 7, 8A, and 8B, we found that while T-NRPCs generally have the ability to influence nerve regeneration (e.g., via neurite outgrowth), it is nevertheless important to further screen T-NRPC samples (e.g., based on human and tonsillar side (the side from which T-NRPCs are obtained via corresponding T-MSCs)) to select and propagate the most effective T-NRPCs.

[0084] NRPCs may also be selected based on their ability to induce neurite outgrowth, which is determined by meeting a threshold for the average number of neurites formed. In particular, the inventors have found that NRPCs can be screened, selected, and proliferated based on their ability to induce neurite outgrowth (e.g., in neuroblastoma cells), and that criteria can be established to evaluate which NRPCs most effectively induce neurite outgrowth. In one embodiment, a given NRPC can induce neurite outgrowth if the average number of neurites formed per neuroblastoma cell in each sample of neuroblastoma cells is at least 15, and the length of the longest neurite formed in each sample of neuroblastoma cells is at least 145 μm. However, in some embodiments, the threshold for the number of neurites formed per neuroblastoma cell may be higher for determining the ability of an NRPC sample to induce neurite growth. For example, the threshold for the number of neurites formed per neuroblastoma cell may be approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, or 100. In some embodiments, the threshold may be a range formed by selecting any two numbers listed in the preceding sentence (for example, the minimum threshold for the number of neurites formed per neuroblastoma cell may be about 12, about 10 to about 30, etc.). In some embodiments, the average number of neurites formed per sample may be determined by averaging the number of neurites formed across multiple samples of neuroblastoma cells. Each sample may be contained in its own well. For example, the number of neuroblastoma cells used in the neurite extension assay (e.g., N1E-115 mouse neuroblastoma cells) may be 1 × 10⁶. 6 cells / ml or 1 × 10⁶ 5 It can be cells / well. In some embodiments, multiple samples (wells) may be used. In one embodiment, 20 to 40 samples (wells) of N1E-115 mouse neuroblastoma cells may be used, or 24 samples may be used via a plate having, for example, 24 wells. NRPC can evaluate the ability to induce neurite growth by measuring the number of neurites formed per neuroblastoma cell with respect to the sample and obtain an average value.

[0085] NRPCs may be further selected based on their ability to induce neurite outgrowth, which is determined by meeting a threshold for the average number of the longest neurites formed. As already discussed, NRPCs may be screened, selected, and proliferated based on their ability to induce neurite outgrowth (e.g., in neuroblastoma cells), and criteria can be established to evaluate which NRPCs most effectively induce neurite outgrowth. In some embodiments, a given NRPC sample may induce neurite outgrowth if the average length of the longest neurites formed in each sample of neuroblastoma cells is at least 145 μm. In some embodiments, the threshold for the length of the longest neurites formed in a sample of neuroblastoma cells (to determine the ability of an NRPC sample to induce neurite growth in a sample of neuroblastoma cells) may be higher. For example, the threshold length for the longest neurite formed in a sample of neuroblastoma cells may be approximately 145 μm, 150 μm, 155 μm, 160 μm, 165 μm, 170 μm, 175 μm, 180 μm, 185 μm, 190 μm, 195 μm, 200 μm, 205 μm, 210 μm, 215 μm, 220 μm, or 225 μm. In some embodiments, the threshold may be a range formed by selecting any two numbers listed in the preceding sentence (for example, the minimum threshold for the average length of the longest neurite formed in a sample of neuroblastoma cells may be approximately 150 μm to approximately 170 μm, etc.). In some embodiments, the average length of the longest neurite formed may be determined by averaging the longest lengths of neurites formed across multiple samples of neuroblastoma cells. In some embodiments, multiple samples (e.g., 20 to 40 samples of neuroblastoma cells) may be used. For example, 24 samples (each contained in a well) may be used via a 24-well plate. Furthermore, 1 × 10⁶ samples per well 5 Individual N1E 115 mouse neuroblastoma cells may be seeded. The NRPC can be evaluated for its ability to induce neurite growth in each sample (well) (for example, by measuring the length of the longest neurite formed).

[0086] Neurite outgrowth between samples containing tonsil-derived MSCs and NRPCs is reduced when CD121a expression is decreased. As previously discussed, neurite outgrowth in precursor or damaged nerve cells (e.g., mouse neuroblastoma N1E115 cell line) may occur more efficiently under certain culture media (e.g., the culture medium in which T-NRPCs are cultured) than under other culture media, based on the active components of such culture media. Such active components may be proteins expressed by NRPCs grown in culture media that influence the induction of neurite outgrowth. The inventors discovered CD121a as a particularly important protein for neurite outgrowth after conducting tests showing that reduced expression of CD121a reduces neurite outgrowth. In these tests, CD121a expression was reduced using small interfering RNA (siRNA). Specifically, the tests included six sample groups: a negative control group, a positive control group, primary Schwann cell samples, T-MSC samples, T-NRPC samples treated with scrambled siRNA, and T-NRPC samples treated with siRNA. Scrambled siRNA served as a negative control, distinguishing the effect on specific genes from nonspecific or cell-related effects. Furthermore, scrambled siRNA helped reduce nonspecific effects and background noise, thus improving the accuracy and reliability of the experiment. Figures 9A and 9B include a series of images 910 showing the results in these sample groups, as well as graphs 920A and 920B showing the number of neurites formed and the length of the longest neurites formed in the sample groups. As shown in Figures 9A and 9B, when CD121a expression was reduced using small interfering RNA (siRNA), neurite growth was significantly reduced between T-MSC and T-NRPC samples. On the one hand, scrambled siRNA functioned to eliminate the effect of siRNA transfection on cells when knocking down the target gene. Consequently, the sample group containing T-NRPCs treated with scrambled siRNA showed a greater number of neurites and longer axonal lengths than the other experimental groups.Therefore, the inventors discovered that the CD121a marker most highly expressed in T-NRPC is an important marker for nerve regeneration, and that reducing CD121a expression by knockdown of CD121a reduces the number of axons and the length of the longest axon.

[0087] CD121a expression varies among different MSC and NRPC samples, as well as across NRPC production stages. Since CD121a has been demonstrated to be important for effective neurite outgrowth, the inventors investigated how CD121a expression differs among different types of MSCs and NRPCs, e.g., MSCs and NRPCs derived from different humans, different regions of humans (including the amygdala), or MSCs and NRPCs obtained from different stages of NRPC production processes. To test CD121a expression, the inventors obtained several sample groups from a working cell bank, including various TMSCs, MSCs, primary Schwann cells, and control groups, and tested CD121a expression (TMSC2001L, NRPC2001L, TMSC2001R, NRPC2001R, TMSC2009L, NRPC2009L, primary Schwann cells, AD-MSC, AD-NRPC, BM-MSC, BM-NRPC, UC-MSC, UC-NRPC). From these samples obtained from the working cell bank stage of the production process, the inventors were able to determine which samples continued to express CD121a at a significant level (e.g., more than 75%) in the product stage. The inventors obtained these selected T-NRPC samples from the product stage of the production process and investigated how CD121a expression increased throughout the production process. These selected T-NRPC samples (T-NRPC2001L, T-NRPC2001R, T-NRPC2009L, and T-NRPC2009R) correspond to T-NRPCs from different humans (humans identifiable by number in the sample names, such as 2001, 2005, 2009, etc.) and / or different tonsillar sides (indicated by "L" for left and "R" for right in the sample names). The results regarding CD121a expression levels are shown in Figures 10A and 10B. Specifically, Graph 1010 in Figure 10A shows the expression levels of the marker CD121a in MSC and NRPC samples obtained from a working cell bank, originating from various regions and humans, while Graph 1020 in Figure 10B shows the expression levels of CD121a among the (final) products for the sample groups T-NRPC2001L, T-NRPC2001R, T-NRPC2009L, and T-NRPC2009R.

[0088] Screening and selecting NRPC candidates with CD121a expression levels of at least 30% significantly improves the quality of NRPCs. Based on the CD121a expression levels shown in Figures 10A and 10B, the inventors discovered that more effective NRPCs (for example, based on the potential for improved neurite outgrowth) can be produced by screening NRPC candidates with high levels of CD121a expression. In particular, the inventors noted that NRPC candidates in a working cell bank (WCB) in which CD121a is expressed at an expression rate of more than 30% can be selected at an earlier stage of the NRPC production process (for example, as MSCs differentiate into their respective NRPCs based on GMP standards). For example, as shown in Graph 1010, the working cell bank (WCB)-derived NRPC candidate groups (T-NRPC2001L, T-NRPC2001R, T-NRPC2009L, and T-NRPC2009R) that had CD121a expression levels above the first expression threshold (i.e., 30%) were ultimately the T-NRPC samples that were able to express CD121a at a level exceeding a higher second threshold expression rate (e.g., 80%) when these samples were in the product stage. Furthermore, these samples were able to maintain a sufficient expression rate (at least 30%) at least while frozen in the product stage. When these samples were not frozen (e.g., after one or more passages after thawing) and / or in the product stage with living standards, the expression rate recovered to an expression level exceeding 80%. Therefore, a CD121a expression level of 30% or higher among NRPC candidate samples at the working cell bank (WCB) stage can be used as a criterion for determining which of these NRPC candidates to select and use in the remaining production process when forming the NRPC product. However, in some embodiments, the threshold for CD121a expression level at the working cell bank stage (e.g., the first threshold) or the threshold for CD121a expression level at the product stage (e.g., the second threshold) may be higher.For example, an NRPC candidate may be selected from the working cell bank if its CD121a expression level exceeds a threshold, i.e., approximately 30%, 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, 70%, 72.5%, 75%, 77.5%, or 80%. In embodiments, the threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC candidate sample may be selected if the CD121a expression rate is at least above a threshold such as approximately 30%, approximately 30% to approximately 80%, or approximately 35% to approximately 50%).

[0089] Frozen state vs. Unfrozen state As described above, the expression level of CD121a appeared to increase significantly when the NRPC and / or NRPC candidate was measured at least once after thawing from a frozen state, compared to when the NRPC and / or NRPC candidate was frozen. Frozen state may refer to the NRPC and / or NRPC candidate being cryopreserved in a suitable medium (e.g., liquid nitrogen), in which case the temperature is below a threshold of approximately -200°C, -195°C, -190°C, -185°C, -180°C, -175°C, -170°C, -165°C, or -160°C. In embodiments, the threshold may be within a range formed by selecting any two numbers (temperatures) listed in the preceding sentence (for example, the NRPC and / or NRPC candidate may be frozen if they are cryopreserved in a suitable medium at a temperature below a threshold such as approximately -180°C, approximately -190°C to approximately -170°C, or approximately -180°C to approximately -160°C). The expression rate or level of a protein marker in a frozen NRPC or NRPC candidate can be determined by measuring the expression rate or level immediately after thawing the NRPC or NRPC candidate. Measuring the expression level immediately after thawing can avoid heat-dependent cellular activities (e.g., enzyme activity) that may affect the expression rate. For example, the time after thawing considered to be immediately after thawing may be about 0.5 seconds, 1 second, 5 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, or less than 1 hour. In embodiments, the time after thawing that forms the upper limit of the time considered to be immediately after thawing may be within the range formed by selecting any two numbers (2) listed in the preceding sentence (for example, the time after thawing that forms the upper limit of the time considered to be immediately after thawing may be less than 1 minute, about 30 seconds to about 30 minutes, less than 1 hour, etc.). In at least one embodiment, the characteristics of the cell immediately after thawing (e.g., the expression level of a given marker protein in an NRPC or NRPC candidate immediately after thawing) may mean the characteristics of the cell before any subsequent passage (e.g., subculturing) after thawing. NRPCs and / or NRPC candidates can be said to be "alive" if they have undergone one or more passages after being thawed from a pre-frozen state.Cells in the master cell bank (MCB) and working cell bank (WCB) stages of the NRPC production process are intended to be frozen unless otherwise specified. Furthermore, cells from the product (F / P) stage (e.g., T-NRPC) are intended to be alive unless otherwise specified.

[0090] Cell thawing process As already discussed, in order to measure characteristics (e.g., the expression level of a given marker protein (e.g., CD121a)) from cells in a frozen state (e.g., working cell bank stage), it may be necessary to perform the measurement immediately after thawing (e.g., before any subsequent one or more passages after thawing). Therefore, measuring the characteristics of frozen cells may involve thawing the cells. Frozen cells (e.g., NRPCs or NRPC candidates in the working cell bank stage) can be thawed by placing the cells in an environment having a predetermined thawing temperature for a predetermined thawing period. The thawing temperature may be approximately 25°C, 30°C, 32.5°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 42.5°C, 45°C, or 50°C. In some embodiments, it may be within a range formed by selecting any two numbers (two temperatures) listed in the preceding sentence (e.g., the thawing temperature may be approximately 36°C to 38°C, approximately 30°C to 45°C, etc.). This thawing period may be approximately 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 4, 4.5, or 5 minutes. In some embodiments, the thawing period may be within a range formed by selecting any two numbers (two times) listed in the preceding sentence (for example, the thawing period may be approximately 2.5 minutes to approximately 3.5 minutes, approximately 3 minutes to approximately 4 minutes, etc.). In at least one embodiment, the thawing temperature is 37°C and the thawing period is 3 minutes.

[0091] CD121a expression levels in T-NRPC are consistently high throughout the entire product stage of passage. Having demonstrated that the product of appropriately selected NRPCs (e.g., T-NRPCs) can express CD121a to a significant level (e.g., at least 80%), the inventors investigated how the expression level of CD121a progresses through various passages at the product stage of the NRPC production process. The inventors obtained subsamples of T-MSCs and NRPCs at each passage from passage 5 to 19 and tested these subsamples for CD121a expression. Figures 11A and 11B show graphs illustrating the CD121a expression levels of T-MSCs and NRPCs at each passage up to the product (Table 1110), the average expression levels of T-MSCs and T-NRPCs at each passage from passage 5 to 19 (Table 1120), and the CD121a expression levels of T-MSCs and NRPCs at each passage up to the product. As shown in Figures 11A and 11B, the high CD121a expression characteristic of T-NRPC (e.g., over 80% in the viable state) is continuously maintained up to passage 19, even after generation. However, CD121a expression in T-MSC samples fluctuates with passage, and the expression level typically decreases.

[0092] CD121a expression levels correlate with neurite outgrowth in the final passage of T-MSCs and T-NRPCs. The inventors also observed that consistently high CD121a expression levels across the entire passage of T-NRPC products corresponded to consistently high neurite elongation in neurite elongation assays prepared using samples of T-NRPC products obtained across the entire passage. The inventors also observed that altered CD121a expression levels across the entire passage of T-MSC products corresponded to altered neurite elongation in neurite growth assays prepared using samples of T-MSC products obtained across the entire passage. The results are shown in Figure 12. Specifically, Figure 12 includes a series of images showing the results of neurite elongation using samples of T-NRPC and T-MSC products obtained across passages P14 to P19. In some embodiments, as described with respect to Figures 14A to 14C, there is a correlation between the neurite elongation assay and the expression levels of the T-MSC and NRPC marker CD121a in each passage up to the product. Furthermore, the inventors evaluated the state of the cells by determining the cell viability and cell size.

[0093] T-NRPC obtained throughout the entire passage process at the product stage can induce high neurite outgrowth. As previously discussed, T-NRPC samples at the product stage can consistently express CD121a at high levels, which correlates with high neurite growth. We demonstrated that high neurite growth is maintained using a neurite growth assay containing T-MSCs and T-NRPCs obtained throughout the entire passage of the product stage. For example, Figures 13A and 13B show neurite elongation observed in a neurite growth assay containing product samples of T-MSCs and T-NRPCs obtained throughout the entire passage. Here, neurite elongation is shown based on the number of axons and the length of the longest axon in each sample. Specifically, Graph 1310 shows the longest neurite length, and Graph 1320 shows the average number of neurites observed in the sample based on T-MSCs obtained throughout the entire passage. Graph 1310 shows the longest neurite length, and Graph 1340 shows the average number of neurites observed in a sample based on T-NRPC obtained throughout the entire passage at the product stage (e.g., after MSCs have fully differentiated into T-NRPCs). In samples based on T-MSCs obtained throughout the entire passage, the number of axons and the length of the longest axon appear to vary. However, in samples based on T-MSCs obtained throughout the entire passage at the product stage, the length of the longest axon and the number of axons are maintained above the threshold, even up to passage 19 after NRPC differentiation. Specifically, as shown in Graph 1330, the length of the longest axon in samples based on T-NRPC products appears to be maintained above the lower threshold of 150 μm, even as late as passage 19. Some samples based on T-NRPC products exceed an even higher threshold of 300 μm. Furthermore, as shown in Graph 1340, the average axonal number in samples based on T-NRPC products appears to remain above the lower threshold of 10, even as late as passage 19. Some samples based on T-NRPC products even exceed the even higher threshold of 20.

[0094] The T-NRPC product was even more effective in CD121a expression after thawing. As previously discussed, the inventors discovered that CD121a is essential for significantly improving nerve regeneration, and that T-NRPC is known to express the CD121a protein particularly well at its product stage. The inventors also discovered that the CD121a expression level is significantly increased at the product stage compared to earlier stages (e.g., the working cell bank stage). The expression rate of CD121a in T-NRPC products was measured after thawing and subculturing the product samples (e.g., after multiple subculturings). Specifically, in this study, the inventors measured CD121a expression in T-NRPC product samples obtained over 15-19 subculturings after thawing the samples (from a frozen state). For comparison, corresponding T-MSC samples that had differentiated into T-NRPC were also obtained and tested for CD121a expression. For consistency, the T-NRPC and T-MSC samples belonged to the human and tonsillar regions corresponding to 2009R (i.e., the samples were T-NRPC2009R and T-MSC2009R). Furthermore, to confirm that the T-MSCs and T-NRPCs were viable for use in treatment after thawing, the inventors also tested the samples obtained after thawing for cellular properties (e.g., cell viability, cell size, and cell population doubling level (cPDL)). Figures 14A to 14C show the results of these tests. Specifically, graphs 1410, 1420, and 1430 show the cell viability, cell size, and cell population doubling level (cPDL) of the T-NRPC2009R product samples compared to the corresponding T-MSC 2009R samples, respectively, obtained throughout pages P15 to P19. Furthermore, Graph 1440 shows the expression of the marker CD121a in the T-NRPC2009R product sample compared to the corresponding T-MSC2009R sample over passages 15–19. As shown in these graphs, the T-MSC and T-NRPC cell samples maintained the characteristics of healthy basal cells throughout each passage. Moreover, the T-NRPC sample maintained high CD121a expression even after thawing.Specifically, an expression level of 70% was observed immediately after thawing (this indicates the possible expression level of the product in the frozen state), but from passage 16 onwards, the expression level recovered to over 80% (this indicates the expression level in the raw, unfrozen state). As previously discussed, the expression level of CD121a between T-MSC samples remained low.

[0095] The method for producing improved NRPCs disclosed herein includes the ability of MSCs to differentiate into NRPCs and the ability of NRPCs to express CD121a. Accordingly, this disclosure describes improved NRPCs for treating and / or regenerating damaged nerves, and methods for generating them. As described through the experiments and tests described above, various embodiments of methods for generating improved NRPCs include using NRPCs differentiated from MSCs obtained from human tonsils expressing the protein markers CD26, CD106, CD112, CD121a, and CD141; selectively growing such T-NRPC candidates that express the protein marker CD121a above a threshold level (e.g., greater than 30%); and selectively growing T-NRPC candidates that exhibit neurite formation. In such embodiments, the ability of MSCs to differentiate into NRPCs is important. Figure 15A shows a series of images 1502–1532 illustrating the differentiation of MSCs from various regions into NRPCs according to exemplary embodiments of this disclosure. This series of images shows that in the process of differentiation into NRPCs, MSCs form neurospheres or free-flowing clusters of neural stem cells. The set of images 1502-1532 confirms that some MSCs are better able to differentiate into NRPCs. For example, AD-MSCs and T-MSCs differentiated well into NRPCs, while BM-MSCs and UC-MSCs did not. Furthermore, as previously discussed, MSCs and NRPCs also differ in their ability to express the important CD121a protein. Figure 15B shows a series of graphs 1540 and 1550 illustrating the expression rates of the marker CD121a among NRPCs derived from various human regions and their corresponding MSCs. The differences in CD121a expression levels confirm that AD-MSCs and T-MSCs are more successfully able to differentiate into NRPCs, while AD-NRPCs ultimately fail to express CD121a, whereas T-NRPCs can express CD121a at significantly higher levels (e.g., 90%). Therefore, among the various NRPCs, T-NRPC expressing CD121a can bring about improvements in the treatment of damaged nerve cells.

[0096] Selecting NRPC candidates that express CD121a above the threshold level is crucial for generating effective NRPC products. As previously discussed, and in relation to Figures 10A and 10B, as shown in Graph 1610 of Figure 16A, the expression level of the marker CD121a varied among MSC and NRPC samples obtained from the working cell bank, and the MSCs and NRPCs originated from various regions. Among these sample MSCs and NRPCs at the working cell bank stage of the NRPC production process (e.g., passage 9), the inventors discovered that selecting samples (NRPC candidates) expressing CD121a above a first expression level threshold (e.g., 30%) resulted in NRPC products expressing CD121a at significantly higher levels in later stages, and therefore significantly effective in nerve regeneration. The selected samples (T-NRPC2001L, T-NRPC2001R, T-NRPC2009L, and T-NRPC2009R) expressed CD121a at least 30% of the first expression level threshold. Furthermore, these selected T-NRPC samples from the working cell bank stage were ultimately able to express CD121a at or above a second expression level threshold (e.g., 80%) when these samples reached the product stage. However, some samples that expressed CD121a levels below the threshold were not selected. Therefore, in some embodiments, a CD121a expression level of 30% or higher among the NRPC candidate samples in the working cell bank (WCB) stage can be used as a criterion (referred to herein as the "first threshold," "first expression level," "first expression rate," or "first expression level threshold") for determining which of these NRPC samples (referred herein as NRPC candidates) to select and use in the remaining production process when forming the NRPC product. However, in some embodiments, the first threshold for CD121a expression level may be higher. For example, an NRPC candidate may be selected if its CD121a expression level exceeds a threshold of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%.In some embodiments, this threshold may be a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (for example, an NRPC candidate sample may be selected if the CD121a expression rate is at least above thresholds such as about 30%, about 30% to about 40%, or about 40% to about 60%). In some embodiments, the product NRPC may further be screened for samples expressing CD121a above a second expression level threshold (e.g., 80% or higher). For example, in some embodiments, a method for producing a valid NRPC may include selecting an NRPC candidate from a working cell bank that satisfies a first threshold for CD121a expression (e.g., 30% or higher), and then, in the product stage, selecting an NRPC from the NRPC that expresses CD121a above a second threshold (e.g., 80% or higher after thawing or after being placed in a living standard). Even at the product stage, the inventors discovered that the CD121a expression rate further increased (for example, up to 90%) as the T-NRPC sample was thawed and subjected to one or more subculturing cycles. Therefore, the expression rates of the NRPC candidates selected from the working cell bank stage and the NRPCs selected from the product are shown next to Graph 1620 in Figure 16B.

[0097] The improved NRPC was tested in mice prepared to simulate critical limb ischemia (CLI). As discussed herein, the improved NRPC described herein was tested in mice simulating severe limb ischemia (CLI) and yielded favorable results. CLI is a debilitating disease in which fat deposits form in the arterial walls of the legs, leading to atherosclerosis and reduced blood flow. As a result, inflammation and necrosis may occur in the tissues of the lower leg. In severe cases, approximately 40% of patients undergo limb amputation, and approximately 20% die within 6 months. To test the efficacy of the improved NRPC disclosed herein, mice were prepared to simulate patients with CLI. Specifically, the femoral artery on the leg side of the mice was ligated and removed to prepare an animal model with the same pathological condition as patients with CLI.

[0098] The experiment to test the effects of improving NPC included several treatment groups and control groups. For this experiment, seven groups of mice were prepared to simulate CLI, and six of them received various treatments (referred to herein as the treatment groups). One of the seven groups prepared to simulate CLI received no treatment and is referred to herein as the negative control group. In addition, one group of mice was normal (i.e., not prepared to simulate CLI) and is referred to as the normal group. Clopidogrel (CP), a treatment for CLI, was selected for one of the six experimental treatment groups. CP was administered in five divided doses of 0.025 mg / 20 g into the leg muscles of mice whose blood vessels had been removed. Four of the six treatment groups of mice received MSCs and NRPCs. Specifically, one treatment group received T-MSCs, another received AD-MSCs, another received T-NRPCs, and another received AD-NRPCs. In two of the treatment groups, the NRPCs administered were differentiated from the respective MSCs administered in those groups. For the remaining treatment group, the mice were given an excipient (CS10). For the treatment groups receiving MSCs and NRPCs, a large number of mice (e.g., 1 × 10⁶) were administered. 6 Each cell in the ) was divided and administered to the muscle regions of the five legs of mice from which blood vessels had been removed. For each experimental group, blood flow to the mouse legs (measured as a percentage of blood perfusion) was analyzed weekly for three weeks. In the group where mice were prepared to simulate CLI, the legs corresponded to legs from which blood vessels had been removed.

[0099] Experimental results in animal models of the clinical lymphoma (CLI) showed that T-NRPC effectively improves blood flow compared to other treatments. Over a period of three weeks (e.g., 21 days), hemoperfusion was measured across the eight groups of the experiment described above, and pathological symptoms of limb necrosis and amputation due to inflammation were determined by visual observation. The experimental results are shown in Figures 17A to 17. Specifically, the set of images 1710 in Figure 17A shows the blood flow analysis over a period of time across all eight groups of animal samples, including seven groups prepared to simulate CLI and six treatment groups receiving various forms of treatment based on MSC, NRPC, CS10, and CP. As shown in the set of images 1710, which show hemoperfusion over 21 days (D0 to D21), blood flow (shown in red) was increased in the group administered with T-NRPC compared to the groups administered with the inducer excipient (CS10), clopidogrel (CP), T-MSC, AD-MSC, and AD-NRPC. Therefore, the inventors found that pathological symptoms caused by lower limb necrosis and the risk of amputation were reduced in the group administered T-NRPC. Figure 17B includes graph 1720 quantifying changes in blood flow over 21 days in all eight groups using a program specifically for laser Doppler imaging. Figure 17C includes graph 1730 showing the pathological conditions (e.g., necrosis, amputation, and survival) observed in the legs of all eight groups of mice on day 21 of the experiment. White bars indicate the number of intact mouse legs (e.g., no necrosis or amputation due to the administered substance); dark gray bars indicate the number of animals whose legs were amputated; and light gray bars indicate the number of animals with necrotic mouse limbs. As shown in graph 1730, the group administered T-NRPC had a higher percentage of intact mouse legs compared to all other groups except the normal group. Thus, the animal studies demonstrate that the improved T-NRPC of this disclosure is effective in treating diseases such as CLI.

[0100] T-NRPC was also effective in suppressing myofibrosis, reducing muscle inflammation, and promoting capillary formation in animal models. The ability of various treatments applied to animal models of CLI to treat myofibrosis, muscle inflammation, and capillary formation over a 21-day course was also examined. As previously discussed, the eight experimental groups of mice obtained from the aforementioned experiments consisted of six treatment groups: a normal group (i.e., mice not prepared to simulate CLI), a negative control group (i.e., mice prepared to simulate CLI but not receiving any treatment), and mice prepared to simulate CLI. These six treatment groups included groups administered CS10, CP, T-MSC, AD-MSC, AD-NRPC, and T-NRPC. Figures 18A and 18B show sets of images illustrating varying degrees of myofibrosis, muscle inflammation, and capillary formation after the treatment period (e.g., on day 21 when treatment was performed in the treatment groups). Specifically, set 1810 shows the results of treatment for myofibrosis, set 1820 shows the results of treatment for muscle inflammation, and set 1830 shows the results of treatment for capillary angiogenesis. As shown in Figures 18A and 18B, the group administered T-NRPC showed less progression of myofibrosis and histological findings that maintained muscle shape. The T-NRPC-administered group also showed the smallest area of ​​inflammation and the most abundant angiogenesis. Therefore, the inventors have discovered that T-NRPC has therapeutic effects in various areas, including anti-inflammatory effects, suppression of myofibrosis, and angiogenesis. Accordingly, the T-NRPC disclosed herein has high therapeutic applicability for severe lower limb ischemic disease.

[0101] Experimental results showed that T-NRPC prevents the progression of myofibrosis. To assess the state of myofibrosis across all eight experimental groups, tissue was taken from the legs of mice and histologically analyzed on day 21 of the experiment (e.g., after a 3-week treatment period). As shown in set 1810, the images of the normal group most closely resembled those of the group treated with T-NRPC, due to the oval or circular distribution of muscle fiber bundles in both images. However, the shape of the muscle fiber bundles in the negative control group showed the common form of myonecrosis, where the muscle fiber bundles were not visible and the shape of the muscle fibers was collapsed. The levels of muscle fiber recovery shown in the groups treated with CS10, CP, T-MSC, and AD-MSC were low.

[0102] Experimental results showed that T-NRPC reduces muscle inflammation. To assess muscle inflammation across all eight experimental groups, tissue was taken from the legs of mice and the degree of inflammatory cell infiltration was analyzed through histological pathology on day 21 of the experiment (e.g., after a 3-week treatment period). As shown in set 1820, areas affected by inflammation were stained a darker shade of purple and infiltrated with inflammatory cells distributed between muscle fibers or muscle masses. No signs of inflammation were observed in the normal group. The negative control group had the highest distribution of inflammatory cells. Groups treated with CS10, CP, T-MSC, AD-MSC, and AD-NRPC showed less inflammation compared to the negative control group, but more inflammation compared to the group treated with T-NRPC.

[0103] Experimental results showed that T-NRPC promotes capillary formation. To evaluate capillary angiogenesis across all eight experimental groups, tissue was taken from the legs of mice and the degree of angiogenesis was analyzed through histological pathology 21 days after experimental administration (e.g., after a 3-week treatment period). As shown in set 1830, since blood vessels contain endothelial cells and erythrocytes, the histological results show that erythrocytes are densely packed in a circular pattern. In the normal group, the largest blood vessel in the mouse leg was the femoral artery. No blood vessels were observed in the negative control group and the CS10-treated group. However, angiogenesis was observed in the CP, T-MSC, AD-MSC, AD-NRPC, and T-NRPC-treated groups.

[0104] T-NRPC also investigated the use of nerve conduction studies in the treatment of Charcot-Marie-Tooth disease. Charcot-Marie-Tooth (CMT) disease is a hereditary disorder affecting 1 in 2,500 people, with heterogeneous phenotypes and genetic causes. CMT1A (CMT1A) is a type of hereditary neurological disorder affecting peripheral nerves and caused by duplication of the peripheral myelin protein 22 (PMP22) gene. The inventors investigated whether the MSCs and NRPCs disclosed herein (e.g., the improved T-NRPC disclosed herein) are effective in treating CMT1A. In one experiment, C22 mice were used. C22 mice are mice that have 7-8 copies of human PMP22 containing approximately 40 kb in the proximal region and are commonly used in the study of Charcot-Marie-Tooth disease. Five randomly selected groups of 5-week-old C22 mice were used in this experiment. Of the five groups, three groups received subcutaneous injections of varying doses of T-NRPC. Specifically, one group received a low dose of T-NRPC (referred to herein as NRPC-L) containing approximately 2.5 × 10^4 NRPC; another group received a medium dose of T-NRPC (referred to herein as NRPC-M) containing approximately 2.5 × 10^5 NRPC; and yet another group received a high dose of T-NRPC (referred to herein as NRPC-H) containing approximately 5 × 10^5 NRPC. Each group received two doses of T-NRPC, with the second dose administered four weeks after the first. Another group received transdermal injections of CS10 (referred to as the "sham" group), and the remaining groups received nothing (referred to as the "wild-type (W / T)" group).

[0105] Higher doses of T-NRPC improved nerve conduction in mice. At week 16, nerve conduction studies were performed to obtain a proximal response to the gastrocnemius muscle (biceps femoris), and histological analysis was performed by tissue excision. Figures 19A to 19C show the results of nerve conduction studies performed on mouse samples, up to graphs 1910, 1920, and 1930. Specifically, graph 1910 shows the amplitude of the waveform related to nerve conduction from the gastrocnemius muscle (biceps femoris). Graph 1920 shows the nerve conduction velocity during nerve conduction from the gastrocnemius muscle (biceps femoris). As shown in graph 1920, nerve conduction velocity was significantly increased in C22 mice implanted with high doses of T-NRPC (e.g., NRPC-H) compared to C22 mice implanted with low doses of T-NRPC or the placebo group. Graph 1930 shows the composite muscle action potential (measured in mV) of compounds related to nerve conduction. Here again, CMAP improvement is observed in C22 mice implanted with high doses of T-NRPC (e.g., NRPC-H) compared to C22 mice implanted with low doses of T-NRPC or a placebo group.

[0106] T-NRPC also increased myelination of neurons in C22 mice. It was also found that administering various levels of T-NRPC to groups of C22 mice increased nerve myelination. Figures 20A–20C show three sets of immunostained sciatic nerve images from five groups of C22 mice from the above experiment, where three of the C22 mouse groups were treated with various levels of T-NRPC (NRPC-H, NRPC-M, and NRPC-L) as described above. Various levels of T-NRPC resulted in various levels of protein markers useful for myelination, as performed by the inventors and predicted by experiments already described herein. The first set of images 2010 shows that C22 mice transplanted with various levels of T-NRPC showed increased expression of proteins that form the periphery of myelin (e.g., MBP, shown in green), which is a precursor of nerve cells (NF-H, shown in red). Furthermore, MBP (green) and NF-H (red) expression increased as the amount of T-NRPC administered to C22 mice increased, indicating that sciatic nerve axon and myelin formation in C22 mice can be improved with T-NRPC.

[0107] T-NRPC also improved muscle regeneration in C22 mice. In this experiment, we also tested the expression of MYH8, a marker protein for regenerating muscle, and laminin, a basement membrane protein of the extracellular matrix, by detecting them from immunochemical staining images. As shown in the second set of images 2020 in Figure 20B, the expression of these proteins was increased in the C22 mouse group injected with T-NRPC. The muscle fiber shape in the T-NRPC-treated group was maintained and restored at a similar level to the W / T group. The third set of images 2030 shows the expression of MYH1E and laminin, both skeletal muscle proteins. Here again, the C22 mouse group treated with NRPC tended to have higher expression of MYH1E and laminin. Furthermore, the muscle fiber shape in the NRPC-H group was improved to a similar degree to that of the W / T group.

[0108] T-NRPC was also found to regenerate myelin sheaths in damaged nerves. It was also found that various levels of T-NRPC administered to groups of C22 mice induced myelin regeneration in nerves (e.g., those with destroyed myelin). Figures 21A–21E show five sets of images illustrating the G ratio and myelination of neurons in five treated C22 mouse sample groups, including three groups treated with various levels of T-NRPC (NRPC-H, NRPC-M, and NRPC-L). In the first set of images, 2110, a cross-section of the mouse sciatic nerve was observed using an electron microscope (TEM) to morphologically observe axonal myelin regeneration induced by administered high, medium, and low concentrations of T-NRPC (×3,000). In the cross-section of the sciatic nerve fiber, a rounded axon and the myelin sheath surrounding the axon can be observed. The second set of images, 2120, shows the results after the treatment has been rendered. As shown throughout both sets of images, samples treated with high concentrations of T-NRPC show axonal myelin regeneration, which is closest to the W / T (normal) group. For example, in the W / T group, normal myelin surrounding the axons can be observed, while in the placebo group, there is little myelin surrounding the axons. Myelin formation surrounding the axons can be observed in groups treated with low, medium, or high concentrations of NRPC. The third set 2130 is a single image illustrating the method of G ratio measurement in electron microscopy nerve fiber images. The fourth set of images 2140 shows that increasing the level of T-NRPC in C22 mice improves the Axon G ratio, as indicated by the increased G ratios shown by the NRPC-L, NRPC-M, and NRPC-H groups, respectively, with the NRPC-H group showing a G ratio value closest to the W / T (normal) group. Image 2150 in the fifth set shows that in C22 mice administered NRPC at low, medium, and high concentrations, respectively, myelin thickness increased with increasing T-NRPC levels. Therefore, these experiments demonstrate that T-NRPC increases myelination in nerve cells and myelin regeneration in damaged myelin.

[0109] T-NRPC was also found to express MPZ while regulating PMP22 to an optimal level. As previously discussed, CMT1A type (CMT1A) is caused by duplication of the peripheral myelin protein 22 (PMP22) gene. PMP22 expression is important for myelin formation during sciatic nerve development, but overexpression of PMP22 can induce CMT1A and demyelinating neuropathy. Therefore, PMP22 needs to be downregulated when overexpressed. On the other hand, myelin protein zero (MPZ) is another protein important for myelin development. Specifically, MPZ is important for the formation and stabilization of the multilayer structure of myelin and is an important marker for Schwann cell development. In a healthy state, it is ideal for PMP22 expression to be proportional to MPZ expression. The inventors investigated whether the T-NRPC of this disclosure has the ability to express and regulate the levels of PMP22 and MPZ in C22 mouse groups administered with various amounts of T-NRPC (NRPC-H, NRPC-M, and NRPC-L). Figures 22A to 22C show three sets of images and graphs illustrating the expression of the markers PMP22 and MPZ in C22 mouse samples. The first set of images, 2210, shows the expression level of PMP22 in red; the second set of images, 2220, shows the expression level of MPZ in green; the third set of images, 2230, is a combination of sets 2210 and 2220, indicating whether the expression of MPZ and PMP22 is in equilibrium; graph 2240 (Figure 22D) shows the PMP22 / MPZ expression ratio for each of the C22 mouse groups.

[0110] The PMP22 / MPZ ratio was 1 in the W / T group. In the set of images 2230, which formed a composite of sets 2210 and 2230, the orange color of the placebo group indicated a high presence of PMP22, in contrast to the yellow color of the W / T group and the T-NRPC-treated group (which indicated a more balanced portion of PMP22 and MPZ). Therefore, these results demonstrate that T-NRPC can effectively promote Schwann cell recovery and downregulation of PMP22 by improving the PMP22 / MPZ ratio to a value similar to that of the W / T (normal) group.

[0111] T-NRPC was able to regulate PMP expression by expressing miR-29a. The inventors also investigated why the NRPC-treated C22 mouse group was able to regulate the overexpression of the PMP22 gene. To confirm the mechanism of regulation of PMP22 expression by T-NRPC, various microRNAs (miRNAs) from exosomes obtained from TMSC and NRPC cultures were analyzed. Figure 23A is a table showing the analysis of several miRNAs to identify those that appeared to be expressed in TMSC and T-NRPC cultures. The inventors found that the expression of a miRNA identified as miR-29a was higher in T-NRPC cultures than in T-MSC cultures. miR-29a is a microRNA that regulates PMP22 expression. Therefore, the inventors found that T-NRPC is effective in treating CMT disease because it can suppress the overexpression of PMP22 via the expression of miR-29a. Therefore, when such T-NRPC was administered to the C22 mouse model, which has the highest incidence of CMT disease, the inventors confirmed that the disease is characterized by overexpression of the PMP22 gene, and that the expression of the PMP22 gene was regulated, resulting in improvement of CMT disease. The inventors found that T-NRPC is characterized by increased expression of miR-29a, and that this increased expression of miR-29a allows T-NRPC to inhibit the expression of the PMP22 gene in C22 mice, thereby treating CMT disease.

[0112] miR-29a expression in T-NRPC As shown in Figures 23A and 23B, the inventors found that T-NRPC (e.g., for use as a product) is characterized by increased miR-29a expression, as is evident from its fold change (f) value of 1.329515 and volume of 1.235058. The fold change (fc) value represents the ratio of the expression of a given microRNA (e.g., miR-29a) in T-NRPC to the expression of the microRNA in the corresponding T-MSC. Therefore, as shown in Figures 23A and 23B, miR-29a is expressed 1.329515 times more in T-NRPC than in T-MSC. Furthermore, this volume represents the ratio of the expression intensity of a given microRNA (e.g., miR-29a) in T-NRPC to the expression intensity of a given microRNA (e.g., miR-29a) in the corresponding T-MSC. Therefore, as shown in Figures 23A and 23B, the expression intensity of miR-29a is 1.235058 times higher in T-NRPCs than in T-MSCs. In some embodiments, T-NRPCs may be selected (for example, to produce the product) based on the fold change (fc) of those T-NRPCs expressing miR-29a (compared to the expression of miR-29a in the respective T-MSCs), such that they at least exceed a threshold of approximately 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, or 1.6. In some embodiments, the threshold may be within a range formed by selecting any two numerical values ​​(2x change values ​​(fcs)) listed in the preceding sentence (for example, T-NRPCs may be selected based on the 2x change values ​​(fc) of those expressing miR-29a that exceed the threshold, such as about 1.1 to about 1.5, about 1.15 to about 1.25). In some embodiments, T-NRPCs may be selected (for example, to produce a product) based on the miR-29a volume of T-NRPCs that at least exceeds the threshold of about 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, or 1.5 (i.e., the ratio of the expression intensity of miR-29 in T-NRPCs to the expression intensity of miR-29a in each T-MSC).In some embodiments, the threshold may be a range formed by selecting any two numerical values ​​(two values) listed in the preceding sentence (for example, T-NRPC may be selected based on volume exceeding the threshold, such as about 1.0 to about 1.5, or about 1.15 to about 1.25). Figures 23A and 23B further show the use of miR-413 as an endogenous control, which may have a constant expression rate and can be used for quantitative comparison of the amount of miR-29a expressed in T-MSC and T-NRPC. For example, as shown in Figures 23A and 23B, the expression rate of miR-29a (measured in fold change (fc) and volume) compared to the expression rate of miR-413 was about four times higher in T-NRPC than in T-MSC.

[0113] The inventors also determined the mean threshold cycle (Ct) value of miR-29a. It is intended that the mean Ct value will be faster when there are more target genes present, and slower when there are fewer target genes. The inventors found that the mean Ct value of miR-29a in T-NRPCs was 18.208, which is faster than the mean Ct value of miR-29a in T-MSCs, and therefore, more miR-29a was found in T-NRPCs.

[0114] T-NRPC is effective in treating any ischemic tissue. The animal studies described above were performed in Balb / c nude mice simulating CLI, but it is intended that the disclosed T-NRPC may provide the disclosed beneficial effects to any ischemic tissue. As is generally known, ischemia refers to a limitation of blood supply to any tissue, muscle group, or organ of the body, resulting in a deficiency of oxygen to the tissue. As a result of ischemia, damaged tissue may result in nerve damage (e.g., demyelination and improper nerve conduction), myofibrosis, muscle inflammation, and reduced blood flow, characteristic of the nerves and tissues shown in Figures 19-23. Therefore, the beneficial effects that T-NRPC delivers to ischemic tissue in the lower limbs of C22 mice may be applicable to other ischemic tissues and / or other animals (e.g., humans).

[0115] Administration and formulation of the compositions described herein The pharmaceutical compositions of this disclosure may be administered orally or parenterally. Specifically, parenterally, they may be administered by, for example, intravenous injection, transdermal injection, subcutaneous injection, intramuscular injection, intravitreous injection, subretinal injection, suprachoroidal injection, ophthalmic injection, intracerebral injection, intrathecal injection, intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisional injection, intracoronary artery injection, intracranial injection, intradural injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection, intraperitoneal injection, intrathoracic injection, intrathecal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intracerebral injection, intravesical injection, subconjunctival injection, intratumoral injection, local injection, etc.

[0116] The dosage of the pharmaceutical composition of this disclosure may vary depending on various factors such as the method of formulation, method of administration, time of administration, route of administration, the response achieved and the degree thereof with respect to the administration of the pharmaceutical composition, the age, weight, overall health status, pathological condition or severity, sex, diet and excretion rate of the subject to whom the pharmaceutical composition is administered, and other drugs or components used together that are well known in the medical field and similar factors. An effective dosage for the desired treatment can be readily determined and prescribed by those skilled in the art.

[0117] The administration routes and methods of the pharmaceutical compositions of this disclosure may be independent of each other, as long as the pharmaceutical compositions can reach the target site, and are not particularly limited.

[0118] Formulation of the compositions described herein for administration to a subject In at least one embodiment, the formulation for the finished product to be administered may be in a frozen state. In some embodiments, the formulation can be thawed before administration according to the thawing process for the cells described above. In other embodiments, the formulation may be administered while in a frozen state. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. For non-aqueous solutions or suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As the base for suppositories, Witepsol, Macrogol, Tween 61, cocoa butter, laurin fat, glycerogelatin, etc. may be used. The pharmaceutical composition of the present disclosure can be prepared into single-dose or multi-dose formulations. In some embodiments, pharmaceutically acceptable carriers and / or excipients can be used in the formulation according to methods that can be easily implemented by those skilled in the art to which the present disclosure pertains. The pharmaceutical composition according to the present disclosure can be prepared into various formulations according to general methods. The composition of the present disclosure may contain one or more known active ingredients having an effect of preventing or treating neurological diseases together with the nerve regeneration-promoting cells derived from stem cells having neuron-regenerating activity.

[0119] Dosage of administration The dosage of the pharmaceutical composition of the present disclosure may vary depending on various factors such as the formulation method, administration method, administration time, administration route, response achieved and its degree by the administration of the pharmaceutical composition, the age, weight, general health condition, pathological condition or severity, gender, diet and excretion rate of the subject to which this pharmaceutical composition is administered, as well as other drugs or components used together and similar factors well-known in the medical field. The dosage effective for the desired treatment can be easily determined and prescribed by those skilled in the art. In at least one embodiment, the dosage is about 1×10 5 cells / kg to about 1×10 7 cells / kg and may include the T-NRPC described herein. In another embodiment, the dosage is such that the T-NRPC described herein is about 1×10 4 cells / kg, 5×10 4 cells / kg, 1×10 5cells / kg, 5×10 5 cells / kg, 1×10 6 cells / kg, 5×10 6 cells / kg, 1×10 7 cells / kg, 5×10 7 cells / kg, or 1 × 10⁶ 8 It may be included in the amount of cells / kg. In some embodiments, the administered dose may contain T-NRPC as described herein within a range formed by selecting any two numbers listed in the preceding sentence (for example, the administered dose contains T-NRPC as described herein, approximately 5 × 10⁻⁶). 4 cells / kg and approximately 5 × 10 7 cells / kg, approximately 1×10 5 cells / kg ~ approx. 1×10 6 (May be included in amounts such as cells / kg). In some embodiments, the amount of T-NRPC as described herein in the administered dose may depend on various factors including the age, weight, and sex of the subject, the disease being treated, and its degree and severity. In some embodiments, the administered dose may contain T-NRPC as described herein as the active ingredient, which may be approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18% of the volume of the administered dose. These are 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, or 92%. In some embodiments, this threshold volume fraction may be within a range formed by selecting any two numbers (two volume fractions) listed in the preceding sentence (for example, the administered dose may have T-NRPC described as the active ingredient with thresholds of at least about 0.2% to about 2%, about 1% to about 15%, and about 2% to about 10%). [Examples]

[0120] Example 1.1 - Acquisition of T-MSC Left and right tonsil tissue from numerous donors obtained from the College of Medicine at Ewha Womans University was isolated and placed in a tube holding 10 mL of DPBS (Dulbeccio phosphate-buffered saline) supplemented with 20 μg / mL gentamicin. The tissue was centrifuged at 1,500 rpm for 5 minutes and then washed twice. The washed tonsil tissue was sliced ​​using sterile scissors.

[0121] To isolate tonsil-derived mesenchymal stem cells from tonsil tissue, the tonsil tissue was incubated in a shaking incubator at 37°C and 200 rpm for 60 minutes after adding an equal weight of enzyme reaction solution. The composition of the enzyme reaction solution is shown in Table 1. TIFF2026516852000001.tif42165

[0122] After adding 5% FBS (fetal bovine serum) to the culture, the mixture was centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the remaining pellet was resuspended in 30 mL of DPBS, then centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the remaining pellet was resuspended in 10 mL of DPBS to prepare the suspension. This suspension was passed through a 100 μm filter. The tonsil-derived mesenchymal stem cells remaining on the filter were washed with 20 mL of DPBS, then centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, ACK lysis buffer was added, and incubation was performed in a 37°C constant temperature water bath for 5 minutes. After adding DPBS to the suspension, centrifugation was performed at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the remaining pellet was resuspended in high-glucose DMEM (10% FBS, 20 μg / mL gentamicin) to prepare the cell suspension. Next, the number of cells in the prepared cell suspension was counted. This cell suspension was seeded into a T175 flask and incubated at 37°C in a 5% CO2 incubator.

[0123] Example 1.2 - Acquisition of AD-MSC Adipose-derived mesenchymal stem cells were purchased from Lonza (human adipose-derived stem cells, catalog number PT-5006, Lonza, Switzerland). The adipose-derived mesenchymal stem cells were cultured using the culture medium provided by Lonza (Bulletkit ADSD, catalog number PT-4505).

[0124] Example 1.3 Measurement of population doubling time As already discussed, population doubling time refers to the time required for the population size to double. After obtaining MSC samples for culturing, the inventors determined cell number data using a flow cytometer. Next, the inventors calculated the population doubling time using the cell number data. Specifically, the inventors used the following formula for doubling time (hours) = (t-t0) × log(2) / log(N) t Based on / N0), in the formula, t is the time at the end of the measurement period, t0 is the time at the start of the measurement period, and N t is the number of cells at time t, and N0 is the initial number of cells at time t0.

[0125] Example 2. Formation of Neurospheres Neurospheres were formed by culturing mesenchymal stem cells from Example 1. Specifically, mesenchymal stem cells were subcultured for 4 to 7 passages. After removing the culture medium, the mesenchymal stem cells were washed with DPBS. The washed cells were treated with TrypLE, and the collected cells were counted. The collected cells were centrifuged, the supernatant was removed, and then they were resuspended in neurosphere-forming medium. The composition of the neurosphere-forming medium is shown in Table 2. TIFF2026516852000002.tif43165

[0126] Neurosphere (1x10) 6Cells resuspended in cell-forming medium were seeded onto an ultra-low adhesion dish (60 mm). The seeded cells were cultured for 3 days at 37°C and 5% CO2. After 3 days of culture, the neurospheres formed on the dish were collected into a 15 mL tube. The collected cells were centrifuged, the supernatant was removed, and a neurosphere suspension was prepared by adding fresh neurosphere-forming medium. This neurosphere suspension was transferred to an ultra-low adhesion dish, and the neurospheres were cultured for 4 days at 37°C and 5% CO2.

[0127] Example 3. Differentiation of candidate nerve regeneration-promoting cells (NRPCs) using neurospheres. The neurospheres formed in Example 2 were finely crushed using a 23-26G syringe needle. The crushed neurospheres were transferred to a 15 mL tube using a pipette and then centrifuged. After removing the supernatant, the crushed neurospheres were resuspended by adding nerve regeneration-promoting cell induction medium to the tube. Various induction media for nerve regeneration-promoting cells include: 1) 5-20% FBS (fetal bovine serum), 2) 5-20 ng / mL bFGF (Peprotech, USA), 3) 100-400 μM butylated hydroxyanisole (Sigma, USA), 4) 5-40 μM folklin (MedCheExpress, USA), 5) 0.1-10% N2 supplement (GIBCO, USA), 6) 1-100 ng / mL brain-derived neurotrophic factor (BDNF, Sigma-Aldrich, USA), 7) 1-100 ng / mL nerve growth factor (NGF, Santa Cruz, USA), and 8) 0.01-1 ng / mL Sonic Hedgehog (SHH, R&D) It was prepared by combining three or more of the following: 9) PDGF-AA (platelet-derived growth factor-AA, Peprotech, USA) in 1-10 ng / mL and 10) DMEM / F12 (containing GlutaMAX) containing 50-300 ng / mL of heregulin-beta 1, Peprotech, USA.

[0128] Neurospheres resuspended in various media were seeded into T175 flasks coated with laminin (2 μg / mL). The seeded neurospheres were cultured for 8-10 days, changing the cell induction medium every 3 days (Figure 1).

[0129] Example 4. Screening of nerve regeneration-promoting cells through analysis of CD marker expression. In tonsil-derived mesenchymal stem cells (T-MSCs), the expression of a total of 242 CD markers was analyzed, and myelin formation was confirmed morphologically between the candidate nerve regeneration-promoting cells of Example 4 and the nerve regeneration-promoting cells differentiated therefrom.

[0130] For CD marker analysis, 3x10 7 Individual target cells were collected. The target cells were washed with DPBS and then centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, washed once with DPBS, and then centrifuged again. The remaining pellet was resuspended in 30 mL of FACS buffer. 100 μL of cell suspension (1 × 10⁶) 5 Cells were seeded into each well of a round-bottom 96-well plate. Then, 10 μL of primary antibody for the CD marker was added to each well of the 96-well plate. After reacting on ice for 30 minutes while blocking light, each well was washed with 100 μL of FACS buffer and then centrifuged at 300 g for 5 minutes. The supernatant was removed, 200 μL of FACS buffer was added to each well, and then centrifuged at 300 g for 5 minutes. A secondary antibody was prepared in FACS buffer at a ratio of 1:200 (1.25 μg / mL). After centrifugation was complete, the supernatant was removed, and then 100 μL of the prepared secondary antibody was added to each well. After reacting on ice for 20-30 minutes while blocking light, each well was washed with 100 μL of FACS buffer and then centrifuged at 300 g for 5 minutes. After removing the supernatant, the target cells were washed by adding 200 μL of FACS buffer to each well. The washing procedure was repeated twice. After washing, the cells were resuspended in 200 μL of FACS buffer in each well, and the expression of the CD marker in the target cells was examined by flow cytometry or FACS (fluorescence-activated cell sorting).

[0131] Figure 3 shows the results of comparing the expression of CD markers in induced neuronal regeneration-promoting cells using a heatmap. As shown in Figure 3, neuronal regeneration-promoting cells (NRPCs) and mesenchymal stem cells (MSCs) showed similar CD marker expression patterns, but differences were observed in the expression patterns of some markers. By comparing the CD marker expression patterns of these MSCs with those of the NRPCs differentiated from these MSCs, we selected CD markers with increased or decreased expression as differentiation markers for nerve regeneration-promoting cells. Figure 3 shows the CD markers with increased or decreased expression in NRPCs.

[0132] As shown in Figure 3, CD markers whose expression was increased in NRPCs (CD106, CD112, CD121a, CD338, etc., compared to the respective MSCs from which the NRPCs originated, and such expression was most pronounced in tonsil-derived NRPCs differentiated from tonsil-derived MSCs (T-NRPCs)). Furthermore, as shown in Figure 3, CD markers whose expression was decreased included CD26, CD54, and CD141. In addition, as shown in Figure 3, the expression of CD121a was particularly high in T-NRPCs compared to other NRPCs. Therefore, as previously discussed, further investigation of the effect of CD121a expression revealed that it is particularly associated with the promotion of neurite outgrowth. Based on the above results, we screened for CD markers whose expression is generally increased or decreased in tonsil-derived neurogenesis-promoting cells (T-NRPCs) derived from T-MSCs. The screened markers are as follows: CD markers whose expression is generally increased: CD106, CD112, and CD121a. CD markers whose expression is generally reduced: CD26 and CD141. The patterns of CD marker expression, which are generally increased or decreased, were similarly observed in pro-neuroproliferative cells differentiated from adipose-derived mesenchymal stem cells in Examples 1-2. Therefore, CD markers (CD26, CD106, CD112, CD121a, and CD141) were found to be useful in identifying NRPCs differentiated from MSCs. While the generally altered expression markers CD121a, CD106, CD112, CD26, and CD141 can be used as differentiation markers for pro-neuroproliferative cells, we found that the high expression rate of CD121a was particularly useful in identifying effective NRPCs, as discussed herein.

[0133] Example 4. Cytokine array assay of nerve regeneration-promoting cells As shown in Figures 4A and 4B, the expression of 507 cytokines was analyzed in T-MSCs, AD-MSCs, and their respective NRPCs (T-NRPCs and AD-NRPCs) differentiated from them. Target cells were cultured for cytokine analysis. Target cells were seeded in flasks and cultured for 3-4 days. When the target cells filled more than 80% of the flask, the culture medium was removed and the target cells were washed twice with DPBS. After washing, the culture medium was replaced with DMEM (Dulbeccio phosphate-buffered saline) which does not contain FBS (fetal bovine serum) or cytokines, in order to eliminate the influence of cytokines. The target cell culture was harvested after 30 hours of culture.

[0134] The collected culture was centrifuged at 3,600 rpm for 30 minutes. The supernatant was transferred to a centrifuge tube equipped with a cellulose membrane and concentrated by centrifugation at 3,600 rpm for 20 minutes. After centrifugation, the acclimatized medium that had passed through the separation membrane was discarded, and the same amount of culture was added. Centrifugation was continued until the volume of the concentrated culture was reduced to 1 mL or less, and the concentrated culture was quantified by the Bradford assay. The concentrated culture was mixed with DMEM to adjust the final concentration to 1 mg / mL.

[0135] A membrane coated with an antibody capable of detecting 507 cytokines (Cytokine Array Kit, RayBiotech, USA) was treated with blocking buffer for 30 minutes. The blocking buffer remaining on the membrane was removed and replaced with concentrated culture material, and the membrane was allowed to react overnight in a refrigerator. The membrane was washed seven times with washing buffer. After adding HRP conjugate streptavidin solution, the membrane was allowed to react at room temperature for 2 hours. After removing the HRP conjugate streptavidin solution, the membrane was washed seven times with washing buffer. After washing, the membrane was immersed in ECL (enhanced chemiluminescence) reagent, and cytokine expression was confirmed using an imaging device.

[0136] The results of comparing cytokine expression in nerve regeneration-promoting cells using heatmaps shown in Figures 4A and 4B. In Figures 4A and 4B, AD-MSC, AD-NRPC, T-MSC, and T-NRPC showed different expression patterns. As previously discussed, cytokines particularly related to nerve regeneration, HGF, μPA, and GRO-α, were expressed more highly in NRPCs than in MSCs, and especially in T-NRPCs.

[0137] Example 5. Screening of candidate cells for nerve regeneration-promoting cells by confirming myelin formation in peripheral nerves. In Example 3, we investigated whether the candidate nerve regeneration-promoting cells prepared had the ability to myelinate peripheral nerves. Specifically, we co-cultured differentiated candidate nerve regeneration-promoting cells with dorsal root ganglia (DRGs) and examined whether myelin formation occurred.

[0138] Dorsal root ganglion (DRG) cells isolated from rats were purchased from Lonza (rat dorsal root ganglion cells, catalog number R-DRG-505, Lonza, Switzerland). Candidate cells were co-cultured with the purchased dorsal root ganglia. DRG cells were cultured using the culture medium provided by Lonza (Primary Neuron Growth Medium Bullet Kit (PNGM), catalog number CC-4461).

[0139] The culture medium was changed every three days. When candidate cells were co-cultured with dorsal root ganglia, myelin formation was confirmed morphologically in some cells (Figures 5-8B). Specifically, as previously discussed, the inventors confirmed that the number of axons and the length of the longest axon were significantly increased in the assay group using T-NRPC in culture medium compared to the assay groups using T-MSC, AD-MSC, or AD-NRPC in culture medium.

[0140] Various changes and modifications to the exemplary embodiments disclosed herein will be obvious to those skilled in the art. It should be understood that various changes and modifications to the exemplary embodiments described herein will be obvious to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of this subject matter and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be included by the appended claims.

Claims

1. Nerve regeneration promoting cells (NRPCs) derived from tonsil-derived mesenchymal stem cells and expressing CD26, CD106, CD112, CD121a, and CD141, wherein CD121a has an expression level of approximately 75% or more.

2. The NRPC according to claim 1, wherein the NRPC is in a passaged state for one or more passages after being thawed from a frozen state, and the expression level of the CD121a is measured in the passaged state.

3. The NRPC according to claim 1, wherein the expression level of CD121 in the NRPC is approximately 90% or higher, the NRPC is in a passaged state for one or more passages after thawing from a frozen state, and the expression level of CD121a is measured in the passaged state.

4. The NRPC according to claim 1, The expression level of CD26 in the aforementioned NRPC is approximately 5% or less. The expression level of CD106 in the aforementioned NRPC is approximately 15% or higher. The expression level of CD112 in the aforementioned NRPC is approximately 50% or higher, and The expression level of CD141 in the aforementioned NRPC is approximately 30% or less. The NRPC is in a thawed state after being thawed from a frozen state without subsequent passage, and the expression levels of CD26, CD106, CD112, and CD141 are measured in the thawed state.

5. The NRPC according to claim 1, The expression level of CD26 in the aforementioned NRPC is approximately 10% to approximately 35%. The expression level of CD106 in the aforementioned NRPC is approximately 10% to approximately 35%. The expression level of CD112 in the aforementioned NRPC is approximately 25% to approximately 90%. The expression level of CD141 in the aforementioned NRPC is approximately 10% to approximately 45%. The NRPC is in a passaged state after being thawed from a frozen state and undergoing one or more passages, and the expression levels of CD26, CD106, CD112, and CD141 are measured in the thawed state.

6. A method for generating an NRPC as described in claim 1, To generate multiple cultures of tonsil-derived mesenchymal stem cells (tonsil-derived MSCs) and form neurospheres; To induce NRPC candidates, multiple cell cultures are generated from the neurospheres; To freeze at least a portion of the aforementioned NRPC candidates; Thawing multiple NRPC candidates from a frozen state; The expression level of CD121a is measured in the state immediately after thawing the aforementioned plurality of NRPC candidates from the frozen state; The method comprising selecting an NRPC from among the plurality of NRPC candidates that expresses CD26, CD106, CD112, CD121a, and CD141, wherein the expression of CD121a is at or above a first expression level, and the first expression level is approximately 30%.

7. The method according to claim 6, wherein each of the plurality of cultures of tonsil-derived MSCs is produced in a separate container, each of which contains a separate culture comprising tonsil-derived MSCs and a culture medium for forming the neurospheres.

8. The method according to claim 7, further comprising: before generating a plurality of cultures of the cells from the neurospheres: To recover the neurospheres from at least a portion of each of the containers containing the neurospheres; The method comprising processing the collected neurospheres to further collect cells from the neurospheres.

9. The method according to claim 8, wherein each of the plurality of cultures of cells derived from neurospheres is produced in a separate container, each of which contains cells collected from neurospheres and a culture medium for inducing the cells into NRPC candidates.

10. The method according to claim 6, wherein the left and right tonsil tissues of one person provide two separate cultures of amygdala-derived MSCs.

11. A method according to claim 6, wherein the plurality of cultures of tonsil-derived MSCs can be produced: To provide the left and right tonsil tissues of one person; To isolate the first tonsil-derived MSC from the left tonsil tissue mentioned above; The method comprising isolating a second tonsil-derived MSC from the right tonsil tissue.

12. The method according to claim 6, further comprising: This includes evaluating whether each of the plurality of NRPC candidates or a subset thereof induces myelin formation on the dorsal root ganglia, The method involves selecting an NRPC that induces myelin formation in the dorsal root ganglia and expresses CD26, CD106, CD112, CD121a, and CD141, wherein the expression of CD121a is at or above a first expression level.

13. A method according to claim 12, wherein the following is evaluated: Co-culturing the dorsal root ganglia and the NRPC candidate to be evaluated; The method further includes examining the dorsal root ganglia and confirming myelin formation thereon.

14. The method according to claim 6, further comprising: The method for evaluating whether each of the plurality of NRPC candidates or a subset thereof induces neurite outgrowth on each sample of neuroblastoma cells includes determining that a given NRPC candidate induces neurite outgrowth if the average number of neurites formed on each neuroblastoma cell in each sample of neuroblastoma cells is at least 15, and the length of the longest neurite formed on each sample of neuroblastoma cells is at least 150 μm; The selection here involves selecting an NRPC candidate that induces neurite outgrowth and expresses CD26, CD106, CD112, CD121a, and CD141, wherein the expression of CD121a is at or above the first expression level.

15. The method according to claim 6, further comprising: The selected NRPC is propagated over multiple passages; To recover NRPC from at least one of the aforementioned multiple passages; The method comprising freezing the collected NRPC.

16. The method according to claim 15, further comprising: The method further includes selecting NRPCs after growth spanning at least one of the aforementioned multiple passages and before harvesting, Here, the further selection is to select the NRPC having an expression level of CD121a at or above the second expression level when measured during one or more passages after thawing. The method wherein the second expression level is 75%.

17. A method for treating damaged nerve cells: The method comprising administering to the body of a subject having the damaged nerve cells a composition containing the NRPC described in claim 1 in an effective amount for inducing myelin formation of the damaged nerve cells or myelin regeneration of Schwann cells.

18. Methods for treating myofibrosis: The method comprising administering to a subject having the myofibrosis a composition containing the NRPC described in claim 1 in an effective amount for treating the myofibrosis.

19. A method for treating muscle inflammation, The method comprising administering to a subject having the muscle inflammation a composition containing NRPC according to claim 1 in an effective amount for treating the muscle inflammation.

20. A method for inducing angiogenesis in ischemic tissue: The method comprising administering to the body of a subject having ischemic tissue a composition containing the NRPC described in claim 1 in an effective amount for inducing angiogenesis.

21. A method for treating critical limb ischemia (CLI), The method comprising injecting into the body of a subject having the CLI a composition containing the NRPC described in claim 1 in an effective amount for treating the CLI.

22. Methods for treating peripheral nerve damage: The method comprising administering to a subject having the peripheral nerve injury a composition containing the NRPC described in claim 1 in an effective amount for treating the peripheral nerve injury.

23. A method for suppressing the overexpression of peripheral myelin protein 22 (PMP22): The method comprises administering a composition containing NRPC according to claim 1 to a local area of ​​the body in which overexpression of PMP22 is confirmed or evaluated, in an effective amount for suppressing overexpression of PMP22 in at least the local area.

24. The method according to claim 23, wherein the method treats the Charcot-Marie-Tooth (CMT) of the subject.

25. The method according to claim 23, wherein the composition is administered immediately after the NRPC is thawed from a frozen state.

26. A method for increasing the expression of miR-29a, wherein the method is: The method comprising administering a composition containing NRPC according to claim 1 to a local region of the body of a subject for whom the need for increased miR-29a expression is confirmed or evaluated, in an effective amount that increases the expression of miR-29a in at least the local region.

27. The method according to claim 26, wherein increasing the expression of miR-29a suppresses the overexpression of peripheral myelin protein 22 (PMP22) in at least a local region.

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