Neuronal regeneration promoting cells (nrpc) and treatment of impaired nerve cells

Abstract

The present disclosure provides novel and innovative compositions of neuronal regeneration promoting cells (NRPCs), methods for producing NRPCs, and methods of treating subjects having impaired nerve cells using NRPCs. In one embodiment, the NRPC is induced by a tonsil-derived mesenchymal stem cell and expresses CD26, CD106, CD112, CD121a, and CD141, where CD121a has an expression level of about 30% or more, which is measured immediately after the NRPC has been thawed from a frozen state. In addition, the NRPC is configured to promote the formation of axons on neuronal cells.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to stem cell-derived neuron regeneration promoting cells (NRPCs), and more particularly to NRPCs having the ability to treat damaged neural cells and methods thereof. BACKGROUND

[0002] Neuronal cells, or neurons, form the structural units of the nervous system and transform and transmit electrical signals. Neurons can include a cell body, dendrites extending from the cell body, and an axon. The axon is a long output structure of the neuron that allows the neuron to propagate electrical signals as action potentials. Some axons are encased in a fatty substance called myelin, which makes the electrical signals propagate more efficiently through the neuron. The myelin acts as a form of insulation for the axon, aiding in sending its signals over long distances. Neuron regeneration promoting cells (NRPCs) can promote the development of myelin around axons or myelination.

[0003] Various neurological conditions can arise from improper or insufficient myelination of neuronal cells. For example, Charcot-Marie-Tooth disease (CMT) is a genetic disease that affects 1 in 2,500 people and is phenotypically and genetically heterogeneous. CMT Type 1A (CMT1A) is a genetic neurological condition that affects peripheral nerves and is caused by a duplication of the gene for peripheral myelin protein 22 (PMP22). Thus, there is a desire and need for effective treatments for CMT, including CMT1A, where the treatment can promote myelination while modulating the overexpression of PMP22.

[0004] Mesenchymal stem cells (MSCs) are commonly used for developing cell therapeutics because they can differentiate into multiple cell types in response to specific stimuli. However, MSCs have difficulty being able to effectively develop into precursor cells for myelinating neurons due to a variety of factors, including low yield or insufficient neurite outgrowth. Thus, there is a desire and need for more effective NRPCs and methods of generating them.

[0005] Various embodiments are presented herein that address one or more of these shortcomings. SUMMARY

[0006] The present inventors have found that conventional methods of preparing neuron regeneration promoting cells (NRPCs) from stem cells are very challenging and the NRPCs formed thereby can not be as effective in treating damaged neural cells. The present disclosure provides new and innovative NRPC compositions, methods for generating NRPCs, and methods of using the NRPCs to treat subjects having damaged neural cells.

[0007] Accordingly, in general embodiments, the present disclosure provides a composition of neuron regeneration promoting cells (NRPCs). The NRPCs are induced from tonsil-derived mesenchymal stem cells. The NRPCs express CD26, CD106, CD112, CD121a, and CD141. CD121a has an expression level of 30% or more.

[0008] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of CD121a in the NRPCs is about 30% to about 50%, and the expression level of CD121a is measured immediately after thawing the NRPCs from a frozen state.

[0009] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of any given protein (e.g., CD121a) is considered to be measured immediately after thawing the NRPCs from a frozen state if the expression level is measured prior to any subsequent passage(s) of the NRPCs after thawing the NRPCs from a frozen state.

[0010] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of CD121a in the NRPCs is about 50% or more, and the expression level of CD121a is measured in one or more passages of the NRPCs after thawing the NRPCs from a frozen state.

[0011] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of CD121a in the NRPCs is about 60% or more, and the expression level of CD121a is measured in one or more passages of the NRPCs after thawing the NRPCs from a frozen state.

[0012] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of CD121a in the NRPCs is about 70% or more, and the expression level of CD121a is measured in one or more passages of the NRPCs after thawing the NRPCs from a frozen state.

[0013] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of CD121a in the NRPCs is about 80% or more, and the expression level of CD121a is measured in one or more passages of the NRPCs after thawing the NRPCs from a frozen state.

[0014] In an aspect of the present disclosure, which can be combined with any other aspect, the expression level of CD121a in the NRPCs is about 90% or more, and the expression level of CD121a is measured in one or more passages of the NRPCs after thawing the NRPCs from a frozen state.

[0015] In an aspect of the disclosure, which can be combined with any other aspect, the expression level of CD26 in the NRPC is 5% or less, the expression level of CD106 in the NRPC is 15% or more, the expression level of CD112 in the NRPC is 50% or more, and the expression level of CD141 in the 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 an aspect of the disclosure, which can be combined with any other aspect, the expression level of CD26 in the NRPC is 10% or more, the expression level of CD106 in the NRPC is 10% or more, wherein the expression level of CD112 in the NRPC is 25% or more, and the expression level of CD141 in the NRPC is 10% or more. The expression levels of CD26, CD106, CD112, and CD141 are measured in one or more passages after thawing the NRPC from a frozen state.

[0017] In an aspect of the disclosure, which can be combined with any other aspect, the expression level of CD26 in the NRPC is 10% to 35%, the expression level of CD106 in the NRPC is 10% to 35%, the expression level of CD112 in the NRPC is 25% to 90%, and / or the expression level of CD141 in the NRPC is 10% to 45%. The expression levels of CD26, CD106, CD112, and CD141 are measured in one or more passages after thawing the NRPC from a frozen state.

[0018] In another embodiment, the disclosure provides a method of producing NRPCs. The method comprises: generating a plurality of tonsil-derived mesenchymal stem cell (tonsil-derived MSC) cultures to form neurospheres; generating a plurality of cell cultures from the neurospheres to induce NRPC candidates; and selecting, among the plurality of NRPC candidates, NRPCs that express CD26, CD106, CD112, CD121a, and CD141 and have a first expression level of about 30% or more for CD121a, the first expression level being measured immediately after thawing the NRPC candidate from a frozen state.

[0019] In an aspect of the disclosure, which can be combined with any other aspect, each of the plurality of tonsil-derived MSC cultures is generated in a separate container, such that each of the containers contains a separate culture comprising tonsil-derived MSCs and a medium for forming neurospheres.

[0020] In an aspect of the disclosure, which can be combined with any other aspect, the method further comprises: collecting neural spheres from each of at least a portion of the containers containing neural spheres; and processing the collected neural spheres to further collect cells from the neural spheres.

[0021] In an aspect of the disclosure, which can be combined with any other aspect, each of the plurality of cell cultures from neural spheres is generated in a separate container, such that each of the containers contains a separate culture comprising collected cells from neural spheres and a culture medium for inducing the cells into NRPC candidates.

[0022] In an aspect of the disclosure, which can be combined with any other aspect, left tonsil tissue and right tonsil tissue of a person provide two separate tonsil-derived MSC cultures.

[0023] In an aspect of the disclosure, which can be combined with any other aspect, the method further comprises: providing a plurality of tonsil-derived MSC cultures comprising: providing left tonsil tissue and right tonsil tissue of a person; isolating first tonsil-derived MSCs from the left tonsil; and isolating second tonsil-derived MSCs from the right tonsil.

[0024] In an aspect of the disclosure, which can be combined with any other aspect, the method further comprises: selecting comprises analyzing expression of CD markers.

[0025] In an aspect of the disclosure, which can be combined with any other aspect, selecting comprises flow cytometry on one or more of CD26, CD106, CD112, CD121a, or CD141.

[0026] In an aspect of the disclosure, which can be combined with any other aspect, the method further comprises: for each of the plurality of NRPC candidates or a subset thereof, assessing whether the NRPC candidate induces myelination on dorsal root ganglia, wherein selecting selects NRPCs that induce myelination on dorsal root ganglia, express CD26, CD106, CD112, CD121a, and CD141, and have a first expression level of about 30% or more for CD121a, the first expression level being measured immediately after thawing the NRPC candidate from a frozen state.

[0027] In an aspect of the disclosure, which can be combined with any other aspect, assessing comprises: co-culturing dorsal root ganglia and the NRPC candidate to be assessed; and subsequently examining the dorsal root ganglia and confirming myelination thereon.

[0028] In an aspect of the disclosure that can be combined with any other aspect, the method further comprises: for each or a subset of the plurality of NRPC candidates, assessing whether the NRPC candidate induces neurite outgrowth on the sample of the corresponding neuroblastoma cell, wherein a given NRPC candidate induces neurite outgrowth if the average number of neurites formed per neuroblastoma cell in the sample of the corresponding neuroblastoma cell is at least 15 and the length of the longest neurite formed in the sample of the corresponding neuroblastoma cell is at least 150 pm; and wherein the selecting further comprises: selecting, among the NRPC candidates, NRPCs that induce neurite outgrowth, express CD26, CD106, CD112, CD121a, and CD141, and have a first expression level of about 30% or more for CD121a, the first expression level being measured immediately after thawing the NRPC candidate from a frozen state.

[0029] In an aspect of the disclosure that can be combined with any other aspect, the method further comprises: expanding the selected NRPCs over multiple passages; and harvesting the NRPCs from at least a portion of the multiple passages.

[0030] In an aspect of the disclosure that can be combined with any other aspect, the method further comprises: discarding at least one NRPC candidate that has a first expression level of less than 30% for CD121a.

[0031] In an aspect of the disclosure that can be combined with any other aspect, the NRPCs are selected that have a second expression level of about 75% or more for CD121a, the second expression level being measured in one or more passages after thawing the NRPC candidate from a frozen state.

[0032] In an aspect of the disclosure that can be combined with any other aspect, the NRPCs are selected that have a second expression level of about 80% or more for CD121a, the second expression level being measured in one or more passages after thawing the NRPC candidate from a frozen state.

[0033] In an aspect of the disclosure that can be combined with any other aspect, the NRPCs are selected that have a second expression level of about 85% or more for CD121a, the second expression level being measured in one or more passages after thawing the NRPC candidate from a frozen state.

[0034] In an aspect of the disclosure that can be combined with any other aspect, the NRPCs are selected that have a second expression level of about 90% or more for CD121a, the second expression level being measured in one or more passages after thawing the NRPC candidate from a frozen state.

[0035] In an aspect of the disclosure, which can be combined with any other aspect, the NRPCs are selected that have a third expression level for CD26 of about 5% or less, the NRPCs are selected that have a fourth expression level for CD106 of about 15% or more, the NRPCs are selected that have a fifth expression level for CD112 of about 50% or more, and the NRPCs are selected that have a sixth expression level for CD141 of about 30% or less. The third, fourth, fifth, and sixth expression levels are measured immediately after thawing the NRPC candidates from a frozen state.

[0036] In an aspect of the disclosure, which can be combined with any other aspect, the NRPCs are selected that have a seventh expression level for CD26 of about 10% or more, the NRPCs are selected that have an eighth expression level for CD106 of about 10% or more, the NRPCs are selected that have a ninth expression level for CD112 of about 25% or more, and the NRPCs are selected that have a tenth expression level for CD141 of about 10% or more. The seventh, eighth, ninth, and tenth expression levels are measured in one or more passages after thawing the NRPC candidates from a frozen state.

[0037] In an aspect of the disclosure, which can be combined with any other aspect, the NRPCs are selected that have a seventh expression level for CD26 of about 10% to about 35%, the NRPCs are selected that have an eighth expression level for CD106 of about 10% to about 35%, the NRPCs are selected that have a ninth expression level for CD112 of about 25% to about 90%, and the NRPCs are selected that have a tenth expression level for CD141 of about 10% to about 45%. The seventh, eighth, ninth, and tenth expression levels are measured in one or more passages after thawing the NRPC candidates from a frozen state.

[0038] In another embodiment, the present disclosure provides a method of treating damaged neural cells, the method comprising: administering any of the compositions comprising the NRPCs described herein to a subject having damaged neural cells in an amount effective to cause myelination of the damaged neural cells or Schwann cell-mediated remyelination.

[0039] In another embodiment, the present disclosure provides a method of treating muscle fibrosis, the method comprising: administering any of the compositions comprising the NRPCs described herein to a subject having muscle fibrosis in an amount effective to treat the muscle fibrosis.

[0040] In another embodiment, the present disclosure provides a method of treating muscle inflammation, the method comprising: administering any of the compositions comprising the NRPCs described herein to a subject having muscle inflammation in an amount effective to treat the muscle inflammation.

[0041] In another embodiment, the present disclosure provides a method of inducing angiogenesis in ischemic tissue, the method comprising: administering any of the compositions comprising the NRPCs described herein to a subject having ischemic tissue in an amount effective for inducing angiogenesis.

[0042] In another embodiment, the present disclosure provides a method of treating critical limb ischemia (CLI), the method comprising: injecting any of the compositions comprising the NRPCs described herein into a subject having CLI in an amount effective for treating CLI.

[0043] In another embodiment, the present disclosure provides a method of treating peripheral nerve injury, the method comprising: administering any of the compositions comprising the NRPCs described herein to a subject having peripheral nerve injury in an amount effective for treating peripheral nerve injury.

[0044] In another embodiment, the present disclosure provides a method of inhibiting overexpression of peripheral myelin protein 22 (PMP22) in a subject. The method comprises: administering a composition comprising the NRPCs described herein to a local area of the subject's body in which overexpression of PMP22 is demonstrated or assessed in an amount effective for inhibiting overexpression of PMP22 at least in the local area.

[0045] In an aspect of the present disclosure, which can be combined with any other aspect, the method treats Charcot-Marie-Tooth disease (CMT) in the subject.

[0046] In an aspect of the present disclosure, which can be combined with any other aspect, a composition comprising the NRPCs described herein is administered to a subject in an effective amount. In one embodiment, the administered composition is in a frozen state. In another embodiment, the composition is administered in one or more passages after the NRPCs are thawed from a frozen state.

[0047] In another embodiment, the present disclosure provides a method of increasing expression of miR-29a. The method comprises: administering a composition comprising the NRPCs described herein to a local area of the subject's body in which a need for increasing expression of miR-29a is demonstrated or assessed in an amount effective for increasing expression of miR-29a at least in the local area.

[0048] In an aspect of the present disclosure, which can be combined with any other aspect, increasing expression of miR-29a results in inhibiting overexpression of peripheral myelin protein 22 (PMP22) at least in the local area.

[0049] In an aspect of the present disclosure, which can be combined with any other aspect, the method treats Charcot-Marie-Tooth disease (CMT) in the subject.

[0050] In the present disclosure, the term "subject" refers to an individual in need of administration of the compositions or neuron regeneration promoting cells of the present disclosure, and includes, but is not limited to, mammals (e.g., humans), birds, reptiles, amphibians, fish, and the like.

[0051] In the present disclosure, "treatment" refers to any effecting of improvement or favorable alteration of symptoms of a disease by administration of the compositions according to the present disclosure.

[0052] Additional features and advantages of the disclosed methods and apparatuses are described in, and will be apparent to, the specifics of the DETAILED DESCRIPTION and the drawings in which it is illustrated. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the drawings and description. Moreover, it should be noted that the language used in the specification is principally intended to be read in a context of readability and guidance rather than to limit the scope of the inventive subject matter. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 Cell size and doubling time of different mesenchymal stem cells (MSCs) derived from different regions according to one exemplary embodiment of the present disclosure are exemplified.

[0054] Figure 2 Cell size, viability, and doubling time of tonsil-derived mesenchymal stem cells (TMSCs) obtained from different humans according to one exemplary embodiment of the present disclosure are exemplified.

[0055] Figure 3 Marker expression rates of MSCs derived from different regions of a human and neuron regeneration promoting cells (NRPCs) differentiated from the MSCs according to one exemplary embodiment of the present disclosure are exemplified.

[0056] Figure 4A And Figure 4B Cell factor expression rates in MSCs and NRPCs derived from different regions of a human according to one exemplary embodiment of the present disclosure are shown.

[0057] Figure 5 is a set of immunofluorescence images showing increased expression of a nerve health-related protein of NRPCs obtained from different stages of the NRPC production process according to one exemplary embodiment of the present disclosure.

[0058] Figure 6A And Figure 6B Different levels of neurite outgrowth in MSCs and NRPCs derived from different regions of a human according to one exemplary embodiment of the present disclosure are shown.

[0059] Figure 7 FIG. 2 is a set of images showing different neurite outgrowth in samples of tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs) according to one example embodiment of the present disclosure.

[0060] Figure 8A and Figure 8B FIG. 3 shows a set of graphs showing the average number of neurites and the average length of neurites for neurite outgrowth assays performed in samples of tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs) according to one example embodiment of the present disclosure.

[0061] Figure 9A and Figure 9B FIG. 4 shows a set of graphs and images showing that neurite outgrowth in T-MSC and T-NRPC samples is reduced when expression of CD121a is reduced using small interfering RNA (siRNA) according to one example embodiment of the present disclosure.

[0062] Figure 10A and Figure 10B FIG. 5 shows expression levels of marker CD121a in working cell banks of MSCs and NRPCs derived from different regions and in NRPCs according to one example embodiment of the present disclosure.

[0063] Figure 11A and Figure 11B FIG. 6 shows expression levels of marker CD121a in T-MSCs and NRPCs through various passages according to one example embodiment of the present disclosure.

[0064] Figure 12 FIG. 7 is a set of images and graphs showing the correlation between neurite outgrowth assays and expression levels of marker CD121a in T-MSCs and NRPCs through various passages according to one example embodiment of the present disclosure.

[0065] Figure 13A and Figure 13B FIG. 8 shows a set of graphs showing the average number of neurites and the average length of neurites for T-MSCs and NRPCs through various passages according to one example embodiment of the present disclosure.

[0066] Figure 14A , Figure 14B and Figure 14C FIG. 9 shows a set of graphs showing expression of CD121a in T-MSCs 2009R and NRPCs 2009R corresponding to passages 15 to 19 according to one example embodiment of the present disclosure.

[0067] Figure 15A and Figure 15B A set of images showing differentiation of MSCs derived from different regions of a human into NRPCs, and a set of graphs showing expression rates of marker CD121a in NRPCs and their corresponding MSCs derived from different regions of a human, are shown in accordance with one exemplary embodiment of the present disclosure.

[0068] Figure 16A and Figure 16B Expression levels of marker CD121a in working cell banks of MSCs and NRPCs derived from different regions, and in NRPCs, are shown in accordance with one exemplary embodiment of the present disclosure.

[0069] Figure 17A , Figure 17B and Figure 17C Analysis of blood flow over time in animal samples having critical limb ischemia (CLI) and undergoing various forms of treatment based on MSCs and NRPCs is shown in accordance with one exemplary embodiment of the present disclosure.

[0070] Figure 18A and Figure 18B A set of images showing muscle fibrosis, muscle inflammation, and capillary formation in animal samples receiving various forms of treatment over time, is shown in accordance with one exemplary embodiment of the present disclosure.

[0071] Figure 19A , Figure 19B and Figure 19C Results of nerve conduction studies performed on mouse samples using different levels of T-NRPCs are shown in accordance with one exemplary embodiment of the present disclosure.

[0072] Figure 20A , Figure 20B and Figure 20C Three sets of images of sciatic nerves immunohistochemically stained showing expression of various protein markers of mouse samples treated with different levels of T-NRPCs are shown in accordance with one exemplary embodiment of the present disclosure.

[0073] Figures 21A-21E Five sets of images indicating G-ratio and myelination of neurons of mouse samples treated with different levels of T-NRPCs are shown in accordance with one exemplary embodiment of the present disclosure.

[0074] Figures 22A-22D Three sets of images and graphs indicating expression of markers PMP22 and MPZ in mouse samples treated with different levels of T-NRPCs are shown in accordance with one exemplary embodiment of the present disclosure.

[0075] Figure 23A and Figure 23B A table illustrating analysis of several miRNAs to determine which miRNAs appear to be expressed in TMSC and T-NRPC cultures is shown, according to one exemplary embodiment of the present disclosure. DETAILED DESCRIPTION

[0076] The presently disclosed subject matter will now be described and discussed in greater detail according to some specific embodiments and examples, some but not all of which are illustrated in the accompanying drawings. Like reference signs refer to like elements or components throughout. The presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the specific embodiments set forth herein. Rather, these embodiments are provided as examples of the presently disclosed subject matter, and only the claims set forth below should be considered to limit the scope of the presently disclosed subject matter. Indeed, the presently disclosed subject matter is subject to many modifications and alternative forms, specific examples of which are provided herein. It should be noted that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. Rather, the claims should be regarded as including any and all embodiments, and equivalents thereof, which are within the scope of the appended claims.

[0077] Various neurological conditions are caused by improper or insufficient myelination Myelin is a lipid-rich substance that surrounds the axons of neuronal cells (e.g., as myelin sheaths) to insulate them and increase the rate of action potential propagation along the axons. Myelin is formed by glial cells such as oligodendrocytes and Schwann cells. Various neurological disorders can result from improper or insufficient myelination of neuronal cells or insufficient growth of axons and other neurites. For example, Charcot-Marie-Tooth disease (CMT) is a genetic disease that affects 1 in 2,500 people and is phenotypically and genetically heterogeneous. CMT type 1A (CMT1A) is a genetic neurological disorder that affects peripheral nerves and is caused by gene duplication of peripheral myelin protein 22 (PMP22).

[0078] Such neurological conditions indicate a need for more effective stem cell therapies Damaged nerves, whether due to improper or insufficient myelination or insufficient neurite growth, can be replaced or regenerated using stem cells. In particular, mesenchymal stem cells (MSCs) are commonly used in the development of cell therapeutics because they can differentiate into multiple cell types in response to specific stimuli. Cell types relevant to restoring damaged nerves can require efficient expression of proteins responsible for replacement, regeneration, and / or myelination of nerves. However, the ability of MSCs to efficiently differentiate into suitable precursor cells (referred to herein as “neuronal regeneration promoting cells (NRPCs)”) and then express proteins responsible for replacement, regeneration, and / or myelination of nerves is made difficult by a variety of factors. For example, the yield of such proteins expressed by NRPCs derived from differentiated MSCs is often low, or the NRPC sample can produce insufficient neurite growth.

[0079] The present disclosure describes new and improved NRPCs for treating damaged neurons and methods for their generation The present disclosure describes more effective NRPCs for treating damaged neurons and methods of generating such NRPCs. The inventors of the present disclosure have discovered that NRPCs differentiated from MSCs obtained from certain regions of a human (e.g., tonsils) appear to have a better rate of expression of relevant proteins for treating damaged nerves of the above-mentioned neurological conditions. Such MSCs can be referred to herein as tonsil-derived MSCs (T-MSCs). Furthermore, the inventors have discovered that NRPCs derived from T-MSCs that express 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 of selectively expanding such T-NRPCs that express such proteins are disclosed. In particular, the inventors have discovered that selectively expanding T-NRPCs that express the protein marker CD121a above a threshold level (e.g., above 30% at the working cell bank (WCB) stage) greatly increases the effectiveness of NRPCs in treating damaged nerves. Furthermore, the inventors have discovered that selectively screening and expanding those T-NRPC samples that exhibit neurite formation results in a more effective product for treating damaged nerves.

[0080] The source of MSCs is relevant to the generation of effective NRPCs As will be discussed herein in connection with the experimental data described herein, the inventors have discovered that the source of MSCs is relevant to generating effective NRPCs. For example, the region of the human body from which the MSCs are harvested has an impact on the ability of the MSCs to efficiently differentiate into NRPCs and / or efficiently express relevant protein markers.

[0081] While MSCs from different regions of a human can express relevant protein markers equally, different MSCs Population doubling times tend to vary For example, Figure 1The population doubling time and cell size of different samples of mesenchymal stem cells (MSCs) derived from different regions of the human body are exemplified according to one exemplary embodiment of the present disclosure. The different regions include tonsil, adipose tissue, bone marrow, and umbilical cord, from which tonsil-derived MSCs (T-MSCs), adipose-derived MSCs (AD-MSCs), bone marrow-derived MSCs (BM-MSCs), and umbilical cord-derived MSCs (UC-MSCs) are obtained, respectively. The efficient generation of NRPCs from MSCs depends on the ability of the MSCs to grow in population size so as to allow a sufficient amount of MSCs to differentiate into desired NRPC candidate cells. The growth in population size of any cell sample can be measured by determining the population doubling time of the cell sample, which is the time (e.g., hours) it takes for the cell count of the cell sample to double. Additionally or alternatively, the growth 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 double during in vitro culture. As shown in Figure 1 the growth appears to be different between the different MSC samples. For example, based on the population doubling time and the population doubling level, it was found that UC-MSCs and T-MSCs have the highest growth in population size. However, regardless of the rate of growth, it was found that the expression levels of characteristic markers that indicate that the cells are MSCs - the markers being CD73, CD90, and CD105 - are approximately the same in each of the four MSC samples. The expression levels confirm the presence of MSCs in the samples taken from the four regions of the human (tonsil, adipose tissue, bone marrow, and umbilical cord). While the expression levels of such proteins confirm that the obtained cell samples do contain MSCs, the expression levels of other proteins indicate that T-MSCs generate more efficient NRPCs than the other MSCs, as will be discussed herein.

[0082] Cell size, cell viability, and population doubling times were measured for MSCs obtained from tonsil regions of different humans.

[0083] As previously discussed, the source of MSCs is relevant to generating effective NRPCs, as the particular region from which MSCs are obtained from a person (e.g., tonsil, adipose tissue, bone marrow, umbilical cord, etc.) can determine the effectiveness of the NRPCs and / or MSCs used to generate the NRPCs. Specifically, as will be described in the following studies, the inventors found that tonsil-derived MSCs are the most effective at generating NRPCs (T-NRPCs) with the best neuroregenerative results (e.g., as measured by neurite outgrowth). To begin the studies, the inventors obtained MSCs from the same region (e.g., tonsil) from different people. However, the inventors tested the obtained MSCs to ensure that they were consistent in cell size, cell viability, and population doubling time. The inventors considered that differences in cell size, cell viability, and population doubling time can affect the effectiveness of the studies (e.g., by introducing unintended variables), and therefore assessed the consistency within the obtained T-MSC samples. Figure 2 Cell size, viability, and doubling time of tonsil-derived mesenchymal stem cells (T-MSCs) obtained from different people were exemplified. The T-MSCs were identified as 2001L, 2001R, 2005R, 2009L, and 2009R, with each designation indicating the person (e.g., by the number 2001, 2005, 2009, etc.) and the left or right tonsil (e.g., L or R) from which the T-MSCs were obtained. Specifically, the population doubling level, cell size, and cell viability of T-MSC samples obtained from at least the people identified as 2001L, 2005R, 2009L, and 2009R were measured over passages. The population doubling level of all T-MSC samples appeared to increase with increasing passage number, but the T-MSC 2009L sample from person 2009L increased at a lower rate. The cell size, as measured by cell diameter in microns, appeared to remain constant for the T-MSC samples. The cell viability, as measured by trypan blue staining, appeared to remain constant for all T-MSC samples. However, there were no significant differences in cell size and viability, as well as population doubling time, at least in the samples at any given passage. Thus, the inventors were able to ensure consistency of the T-MSC samples obtained from the people.

[0084] Use of passaging in the methods described herein As used herein, passaging can indicate a process in which a cell culture from a sample is subcultured, i.e., harvested and reseeded into one or more ‘daughter’ cell culture flasks, so that the reseeded cells develop into a cell culture again in their respective daughter cell culture flasks. This process can be repeated such that the cell culture from the ‘daughter’ cell culture flasks can be subcultured again in subsequent ‘daughter’ cell culture flasks (or other suitable containers). The number of passages can indicate the number of these iterations. For example, passage 5 (P5) indicates that cells from an existing cell culture have been harvested and reseeded into new cell culture flasks (to develop into a new cell culture again) five times. As described herein, the methods of producing the disclosed NRPCs for treating damaged nerves can include multiple culturing and subculturing of cell samples (e.g., of MSCs, candidate NRPCs, and NRPCs). By doing so, each subsequent cell culture is supplemented with fresh growth medium, thereby optimizing the expansion, health, and stability of the cell culture.

[0085] Expression rates of CD121a vary depending on the source of MSCs As previously discussed, the inventors discovered that by selectively screening for NRPCs expressing certain marker proteins, in particular CD121a, new and improved NRPCs for treating damaged cells can be generated. The inventors also discovered that the rate of expression of CD121a varies depending on the source of the MSCs differentiated into NRPCs. Figure 3Marker expression rates of MSCs derived from different regions of a human and NRPCs differentiated from the MSCs according to one exemplary embodiment of the present disclosure are exemplified. The expression rates are exemplified by a heatmap 310, a table 320, and a histogram 330. The heatmap 310 indicates the expression rates of various marker proteins, including CD121a, in relation to the treatment of damaged neurons with intensity of red color. The heatmap 310 confirms that the expression rates of the marker proteins are different according to the sources of the MSC samples, and the marker expression rates are also different between the differentiated NRPC samples. The table 320 lists the actual expression rates of various marker proteins in relation to the treatment of damaged neuronal cells in the MSC and NRPC samples, and the histogram 330 exemplifies these actual expression rates (using the height of the bars) that vary according to the sources of the samples. In particular, the table 320 and the histogram 330 show that the expression rate of CD121a in T-MSCs is higher than that in MSCs derived from other tissues (AD-MSCs, BM-MSCs, and UC-MSCs). Furthermore, the table 320 and the histogram 330 show that the expression rate of CD121a in T-NRPCs is much higher than that in AD-NRPCs. The inventors have found that by selectively screening, culturing, and expanding MSCs and / or T-NRPCs based on selection criteria defined by the expression rates of the relevant marker proteins, particularly CD121a, the resulting T-NRPC products are significantly more effective in treating damaged neuronal cells. Based on the experimental results shown, Figure 3 The expression rate of CD121a is also different in each MSC sample. The expression rate of CD121a in T-MSCs is higher than that of the other MSCs. When MSCs of each region are differentiated into their corresponding NRPCs, the expression rates similarly increase, but the rate of increase varies according to the sources of the MSCs. In the case of AD-MSCs derived from adipose tissue, an increase in the expression rate from 1% (in AD-MSCs) to 17% (in AD-NRPCs) is observed, but the increase in the expression rate is significantly lower than that of T-MSCs derived from tonsils, in which the expression rate increases from 37% (in T-MSCs) to 91% (in T-NRPCs). The experimental results show that T-NRPCs can be identified and selected based on a high expression rate of CD121a.

[0086] Respectively, selectable and non-selectable samples based on expression rates of protein markers that do or do not meet expression rate thresholds Selectivity can depend on a first threshold of CD121a expression at an early stage in the NRPC production process As discussed herein, a cell sample (e.g., an NPRC and / or an NPRC candidate) can or can not be selected (e.g., deemed “selectable”) based on the expression rate of a protein marker of interest of the cell sample. Depending on the protein marker, different threshold values can be established for the expression rate of the protein marker. If the expression rate of a given protein marker for a given cell sample is higher than the threshold expression rate specified for the given protein marker, the cell sample is deemed a selectable cell sample. However, if the expression rate of a given protein marker for a given cell sample is lower than the threshold expression rate specified for the given protein marker, the cell sample is deemed a non-selectable cell sample. Further, the ability of a cell to express a protein can depend on the state of the cell when measuring the protein expression level.

[0087] For example, a cell can be in a frozen state, and the expression level of a protein in a cell in a frozen state can be determined and / or inferred by measuring the expression level of the protein in the cell immediately after the cell is thawed from the frozen state. By measuring the expression level immediately after thawing, heat-dependent cellular activities (e.g., enzymatic activities) that can affect the expression rate can be avoided. The duration of time after thawing that can be considered “immediately after thawing” can be within about 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 time after thawing that forms an upper limit of what is considered immediately after thawing can be within a range formed by selecting any two numbers (two times) listed in the preceding sentence (e.g., the time after thawing that forms an upper limit of what is considered immediately after thawing can be about 1 minute to about 5 minutes, about 30 seconds to about 10 minutes, about 2 minutes to about 4 minutes, etc.). Additionally or alternatively, the time that a cell is “immediately after thawing” can be prior to any passaging after the cell is thawed. In embodiments, the threshold for a selectable cell sample can be higher for a cell in a non-frozen or “live” state (e.g., as a product) than for a cell in a frozen state based on the expression rate or level of a protein marker expression. A cell in a non-frozen or “live” state can include a cell that has been passaged one or more times after being thawed from a frozen state. It is contemplated that other protein markers can have different (e.g., lower or higher) threshold expression rates to determine whether a cell expressing such a protein marker is selectable or non-selectable. In some embodiments, a cell in a non-frozen or live state can not have previously been in a frozen state, and thus can not require any thawing process prior to any one or more passagings prior to measuring any protein expression rates.

[0088] Selectivity can also depend on a second threshold of CD121a expression at a later stage in the NRPC production process For example, in at least some embodiments, a cell sample can be selected (e.g., deemed “selectable”) if the cell sample expresses CD121a at an expression rate that is higher than a first threshold (e.g., 30%) when the cell is in a frozen state (e.g., during a working cell bank stage), but at an expression rate that is higher than a second threshold (e.g., 80%) when the cell is in a non-frozen or live state (e.g., during a product stage). The different thresholds of protein expression required to select a cell sample can depend on the stage at which the cell sample is in the NRPC production process, such that if the cell sample is in an early stage, the protein expression can require a lower threshold, but if the cell sample is in a later stage, the protein expression can require a higher threshold. In particular, a lower threshold of CD121a expression can be set to select and expand cell samples in a working cell bank stage. The cell samples selected in this early stage (referred to herein as “NRPC candidates”) can be in a frozen state, and can comprise MSCs in the process of differentiating into corresponding NRPCs. Additionally or alternatively, the NRPC candidates can already be differentiated into NRPCs, but can require further development and / or differentiation. For example, the inventors found that NRPC candidates having a CD121a expression level that is higher than a first threshold rate of about 30% in a working cell bank stage were able to ultimately differentiate into desirable NRPC samples that were able to express CD121a above a second threshold in a later stage (e.g., a product stage). However, in some embodiments, the first threshold of CD121a expression level in this early stage can be higher or lower. For example, NRPC candidates can be selected if their CD121a expression level is at least higher than a threshold of about 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 can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., NRPC candidate samples can be selected if their CD121a expression rate is at least higher than a threshold of about 30%, about 30% to about 40%, about 35% to about 50%, etc.).

[0089] Some MSCs and NRPCs are non-selectable because they do not meet the expression rate threshold for CD121a A second threshold of CD121a expression can be used to select from products of NRPCs, which, as previously discussed, can be in a live or non-frozen state. For example, the inventors found that NRPC candidates with CD121a expression levels above a second threshold rate of about 80% in the product stage (one or more passages after thawing) were able to ultimately form improved NRPCs that provide the advantages described herein. However, in some embodiments, the second threshold of CD121a expression levels at this later stage can be higher or lower. For example, for NRPCs in a later stage (e.g., a product stage and / or a “live” state), the NRPCs can be selectable if they express CD121a at an expression rate that is at least higher than a second threshold of about 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In embodiments, the threshold can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., a cell is selectable if it expresses CD121a at an expression rate that is at least higher than a threshold of about 70%, about 72.5% to about 77.5%, about 85% to about 95%, etc.).

[0090] Figure 3 Figure 3 Expression rates of various protein markers, including CD121a, are shown for MSCs and NRPCs. As shown in Figure 3 some cell samples, the expression rate of CD121a increased as the cell sample differentiated from MSCs to NRPCs. For example, the expression rate of CD121a was higher for adipose tissue-derived NRPCs (AD-NRPCs) than for adipose-derived MSCs (AD-MSCs), from which the former were differentiated. However, despite the increase, the CD121a expression rate was not sufficient for the AD-MSCs or AD-NRPCs to be considered selectable. As Expression rates of other proteins - CD26, CD106, CD112, and CD141 - are also relevantCD121a protein marker at a sufficiently high expression rate (e.g., above about 30%), and thus are not selectable. In contrast, CD121a expression in tonsil-derived MSCs (T-MSCs) is higher than other tissue-derived MSCs. The inventors found that T-MSC samples are able to satisfy the first threshold (e.g., about 30%) by expressing CD121a at 37.04%, and thus are considered selectable. The inventors also found that CD121a expression rate increases as T-MSCs differentiate into their corresponding NRPCs (T-NRPCs). After selectively differentiating T-MSC samples into their corresponding T-NRPCs, samples of live T-NRPCs obtained at the product stage (e.g., after the first subculture (e.g., passaging) after thawing) are also able to successfully express the CD121a protein marker by expressing CD121a at 91.26%, which is above the second threshold (e.g., about 80%), and thus are also considered selectable.

[0091] Figure 3 Notwithstanding the observation that T-NRPCs express CD121a at a significantly high level, Figure 3 Other relevant trends in expression rates of proteins are also shown when different MSCs differentiate into their corresponding NRPCs. For example, when comparing the protein marker expression patterns of MSCs to the protein marker expression patterns of the corresponding NRPCs that the MSCs differentiate into, certain CD markers are associated with a significant increase or decrease in expression rate as the MSCs differentiate into their corresponding NRPCs. As shown in Table 320 and histogram 330, Figure 3 The CD markers that increase in expression in NRPCs (when compared to the corresponding MSCs of NRPC origin) include CD106 and CD112 (in addition to CD121a described above). The increase in expression is most significant for tonsil-derived NRPCs (T-NRPCs) that differentiate from tonsil-derived MSCs (T-MSCs). Further, as shown in Table 320 and histogram 330, When NRPCs or NRPC candidates are in a product stage, NRPCs can be selected based on expression levels of CD106, CD112, and CD121aAs shown, CD markers that are reduced in expression in NRPCs (when compared to corresponding MSCs of NRPC origin) include CD26 and CD141. This pattern of increased (e.g., CD106, CD112, and CD121a) or reduced (CD26 and CD141) expression of CD markers can thus be used to identify NRPCs differentiated from MSCs. As discussed herein, while markers CD121a, CD106, CD112, CD26, and CD141 (expression of which generally changes) can be used as differentiation markers for neuron regeneration promoting cells, the significantly high expression rate of CD121a is particularly useful for identifying effective NRPCs.

[0092] When NRPCs or NRPC candidates are in their working cell bank stage, NRPCs can be selected based on expression levels of CD106, CD112, and CD121a When NRPCs or NRPC candidates are in their product stage, NRPCs can be selected based on expression levels of CD26 and CD141 An increased expression rate of CD106, CD112, and CD121a, and a reduced expression rate of CD26 and CD141 can be identified to select NRPCs differentiated from MSCs. As discussed herein, while markers CD121a, CD106, CD112, CD26, and CD141 (expression of which generally changes) can be used as differentiation markers for neuron regeneration promoting cells, the significantly high expression rate of CD121a is particularly useful for identifying effective NRPCs. Figure 3As shown by the specific examples, based on the measurement of the expression level of the above proteins of T-NRPC when the product stage is in a live non-frozen state (e.g., after one passage post-thaw), T-NRPC expresses CD106 at 20.13%, CD112 at 59.93%, and CD121a at 91.26%. In some embodiments, the NRPC can be selected based on the expression rate of CD106 being at least above a threshold value of about 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, 35%, 37.5%, or 40%. In embodiments, the threshold value can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., the NRPC can be selected if the expression rate of CD106 is at least above about 10%, about 10% to about 30%, about 15% to about 25%, etc.). In some embodiments, the NRPC can be selected based on the expression rate of CD112 being at least above a threshold value of about 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%. In embodiments, the threshold value can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., the NRPC can be selected if the expression rate of CD112 is at least above about 25%, about 25% to about 40%, about 30% to about 50%, etc.). As previously discussed, the effective T-NRPC can be identified and selected based on a high expression rate of CD121a. For example, the T-NRPC can be identified and selected based on the expression rate of CD121a being at least above a threshold value while the T-NRPC is in a non-frozen state (e.g., after one or more passages post-thaw). The threshold value can be about 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 100%. In embodiments, the threshold value can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., the T-NRPC can be identified and selected when expressing CD121a at least above a threshold value, wherein the threshold value is about 80%, about 82.5% to about 87.5%, about 85% to about 95%, etc.).

[0093] When NRPCs or NRPC candidates are in their working cell bank stage, NRPCs can be selected based on expression levels of CD26 and CD141 NRPCs can be selected based on characteristic expression patterns of CD26 and CD141 In some embodiments, T-NRPCs can be selected based on expression rates when the T-NRPCs or T-NRPC candidates are in a working cell bank stage (e.g., immediately after thawing from a frozen state (e.g., prior to any subsequent passaging after thawing)). As previously discussed, the expression rate of a protein in a cell in a frozen state can be determined and / or inferred by measuring the expression rate of the protein in the cell immediately after thawing the cell from a frozen state. By measuring the expression rate immediately after thawing, heat-dependent cellular activities (e.g., enzymatic activities) that can affect the expression rate can be avoided. The duration of time after thawing that can be considered “immediately after thawing” can be within about 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 upper limit of the time after thawing that is considered immediately after thawing can be within a range formed by selecting any two numbers (two times) listed in the preceding sentence (e.g., the upper limit of the time after thawing that is considered immediately after thawing can 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 can be identified and selected based on an expression rate of CD106 that is at least above a threshold value, which can be about 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, or 35%, when the T-NRPCs or T-NRPC candidates are in a working cell bank stage. T-NRPCs can be identified and selected based on an expression rate of CD112 that is at least below a threshold value, which can be about 50%, 52.5%, 55%, 57.5%, 60%, 62.5%, 65%, 67.5%, or 70%, when the T-NRPCs or T-NRPC candidates are in a working cell bank stage. As previously discussed, T-NRPCs or T-NRPC candidates can also be selected based on a threshold expression level of CD121a when the T-NRPCs or T-NRPC candidates are in a working cell bank stage. For example, T-NRPCs or T-NRPC candidates can be selected if their CD121a expression level is at least above a threshold value, which can be about 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 can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., if the expression rate of CD121a is at least higher than a threshold of about 30%, about 30% to about 40%, about 35% to about 50%, etc., then the NRPC candidate sample can be selected).

[0094] Figure 3 T-NRPCs were found to express neuroregeneration-related cytokines at significantly higher levels than other NRPCs When the T-NRPC is in its product phase (e.g., in a live, unfrozen state after one or more passages following thawing), the T-NRPC can be selected based on the expression level of CD26 and CD141. For example, in some embodiments, the T-NRPC can be selected based on the expression rate of CD26 being at least higher than a threshold of 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%. Figure 4A In the specific experiment shown, the T-NRPC was selected based on the expression rate of CD26 being 21.77% and the expression rate of CD141 being 23.75%.

[0095] In some embodiments, the T-NRPC can be identified and selected based on the expression rate of CD26 being at least higher than a threshold of about 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 can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., if the expression rate of CD26 is at least higher than about 10%, about 10% to about 20%, about 15% to about 25%, etc., then the NRPC can be selected).

[0096] In some embodiments, the T-NRPC can be identified and selected based on the expression rate of CD141 being at least higher than a threshold of 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 embodiments, the threshold can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., if the expression rate of CD141 is at least higher than about 10%, about 10% to about 20%, about 15% to about 25%, etc., then the NRPC can be selected).

[0097] Figure 4A Figure 4B In some embodiments, T-NRPCs or T-NRPC candidates can be selected based on expression rates when they are at the working cell bank (WCB) stage (e.g., immediately after thawing from a frozen state (e.g., prior to any subsequent passaging after thawing)).

[0098] At the WCB stage, T-NRPCs can be identified and selected when the expression rate of CD26 is less than 5%. T-NRPCs can be identified and selected based on an expression rate of CD26 that is at least below a threshold value, which can be about 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 can be identified and selected when the expression rate of CD141 is less than 30%. T-NRPCs can be identified and selected based on an expression rate of CD141 that is at least below a threshold value, which can be about 1%, 5%, 10%, 15%, 20%, 25%, 28%, 28.5%, 29%, 29.5%, or 30%. In embodiments, T-NRPCs can be identified and selected when the expression rate of CD26 is less than 5% and when the expression rate of CD141 is less than 30%.

[0099] In embodiments, T-NRPCs can be selected based on a threshold value that is different from the examples described above. In certain embodiments, T-NRPCs can be identified and selected based on an expression rate of CD26 that is at least below a threshold value, which can be about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 32.5%, or 35%, the expression rate of CD26 being measured when the T-NRPC or T-NRPC candidate is at the working cell bank stage. In embodiments, T-NRPCs can be identified and selected based on an expression rate of CD141 that is at least below a threshold value, which can be about 32.5%, 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, or 50%, the expression rate of CD141 being measured when the T-NRPC or T-NRPC candidate is at the working cell bank stage.

[0100] By co-culturing NRPCs with dorsal root ganglion (DRG) neurons, NRPCs can be used for myelination and to improve the stability of nerves Additionally or alternatively, T-NRPCs can be identified based on the difference in expression rates of CD26 and CD141 by the NRPCs when compared to the expression rates of CD26 and CD141 by the corresponding MSCs. That is, the desired expression pattern of the NRPCs (e.g., T-NRPCs) can show reduced expression rates when compared to the MSCs from which the NRPCs were differentiated. For example, in Based on expression of MBP in dorsal root ganglion, the ability of T-NRPCs to induce myelination can be assessedIn the specific example shown, the expression rate of CD26 decreased from 48.48% for T-MSCs to 21.77% for T-NRPCs (a decrease of 26.71%), and the expression rate of CD141 decreased from 60.36% for T-MSCs to 23.75% for T-NRPCs (a decrease of 36.61%). In contrast, the expression rate of CD26 decreased from 90.03% for AD-MSCs to 27.98% for AD-NRPCs (a decrease of 62.05%), while the expression rate of CD141 actually increased from 36.95% for AD-MSCs to 44.96% for AD-NRPCs (an increase of 8.01%). In some embodiments, T-NRPCs can be identified and selected based on a decrease in CD26 expression rate (i.e., delta) between the CD26 expression rate of the NRPC and the CD26 expression rate of the corresponding MSC. The decrease in CD26 expression rate can be at least above a threshold, which can be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In embodiments, the threshold can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., an NRPC can be selected if the decrease in CD26 expression rate between the CD26 expression rate of the NRPC and the CD26 expression rate of the corresponding MSC is at least about 10%, about 10% to about 20%, about 15% to about 25%, etc.). In some embodiments, T-NRPCs can be identified and selected based on a decrease in CD141 expression rate (i.e., delta) between the CD141 expression rate of the NRPC and the CD141 expression rate of the corresponding MSC. The decrease in CD141 expression rate can be at least above a threshold, which can be about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%. In embodiments, the threshold can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., an NRPC can be selected if the decrease in CD141 expression rate between the CD141 expression rate of the NRPC and the CD141 expression rate of the corresponding MSC is at least above about 20%, about 20% to about 30%, about 25% to about 45%, etc.).

[0101] NRPCs are able to more effectively promote myelination when co-cultured with DRG neurons because they undergo the various stages of NRPC production Cytokines are small proteins that are important in cell signaling. In particular, various cytokines are known to facilitate signaling between NRPCs and other cells to treat damaged neural cells. Such cytokines include: hepatocyte growth factor (HGF), urokinase-type plasminogen activator (uPA), and growth-regulated oncogene-alpha (GRO-alpha). To provide more effective NRPCs in treating damaged neural cells, it would be useful to screen NRPCs for their ability to express such cytokines at sufficient expression rates. The inventors tested the ability of MSCs derived from different regions of a human (T-MSCs derived from tonsils and AD-MSCs derived from adipose tissue) and corresponding NRPCs derived from the MSCs (T-NRPCs and AD-NRPCs) to sufficiently express such cytokines - HGF, uPA, and GRO-alpha. The expression rates were compared to a control group (primary Schwann cells) shown in heat map 410 as "Primary Schwann Cells." Figure 5 Figure 5 Figure 5 Cytokine expression rates in MSCs and NRPCs derived from different regions are illustrated via heat map 410 and graphs 420A-C according to one example embodiment of the present disclosure. The inventors found that T-NRPCs were discovered to express cytokines related to neural regeneration at significantly higher levels than other NRPCs differentiated from MSCs from other regions of a human. For both the adipose tissue-derived sample group and the tonsil-derived sample group (e.g., MSCs and NRPCs), the NRPC samples had higher expression of the aforementioned cytokines than the corresponding MSC samples from which the NRPC samples were differentiated. For example, the T-NRPC sample was the only sample among the other samples (AD-MSC, AD-NRPC, and T-MSC samples) to express the HGF cytokine. Although the AD-NRPC sample expressed uPA at a higher rate than the AD-MSC sample (i.e., 300%), the T-NRPC sample expressed uPA at a much higher rate than any of the remaining samples, including the T-MSC sample (i.e., 1400%). Furthermore, although the AD-MSC sample expressed GRO-alpha (i.e., intensity of 5000%), the T-MSC sample did not express GRO-alpha at a significant rate, the T-NPC expressed GRO-alpha at a higher rate than any of the samples, at 7000%. Thus, based on the expression rates of cytokines related to neural regeneration, the inventors found that T-NRPCs are a more optimal choice for NRPCs to treat damaged neural cells than AD-NRPCs.

[0102] ​ ​ ​​Dorsal root ganglion (DRG) neurons are a cluster of neurons in the dorsal root of the spinal nerve. It has been discovered that when NRPCs are co-cultured with dorsal root ganglion (DRG) neurons, the NRPCs can be induced to transdifferentiate into Schwann cell-like cells that can myelinate (e.g., ensheath) the axons of the DRG neurons. As previously discussed, myelination around axons insulates the axons and allows for more efficient long distance propagation of electrical signals through neurons. Thus, by promoting myelination in DRG neurons, NPRCs can treat damaged neural cells by myelinating the damaged neurons and / or by forming new myelinated neurons.

[0103] ​ Based on these experiments, it was discovered that the ability of NRPC candidates to induce myelination can be assessed 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 the NRPCs and / or NRPC candidates to induce myelination can be assessed by subsequently examining the dorsal root ganglion and confirming myelination based on the expression of MBP protein in the sample containing the dorsal root ganglion. In some embodiments, the expression of MBP protein can be detected by immunofluorescence, e.g., after performing immunofluorescence staining and examining for immunofluorescence sensitivity. Because the generation, development, and myelination of nerve fibers and other feeder cells from dorsal root ganglion is affected by cell culture conditions, co-culturing dorsal root ganglion with NRPC candidate samples can be used to assess the ability of each NRPC candidate sample to myelinate feeder cells of the dorsal root ganglion. Although dorsal root ganglion is used in this experiment, because dorsal root ganglion is known to elicit the generation, development, and myelination of nerve fibers and other feeder cells at least in humans, it is expected that cells equivalent to dorsal root ganglion found in other species can be used to evaluate the ability of NRPC candidate samples to induce myelination in other species.

[0104] ​ ​ NRPCs are able to differentiate and myelinate the axons of DRG neurons, or otherwise increase the stability of DRG neurons by expressing relevant proteins such as MBP and TuJl. In particular, MBP is important in the process of myelination of nerves, while TuJl contributes to the stability of the nerve cell body and microtubules. However, the inventors found that the rate of expression of such proteins increased with further increases in the NRPCs in their production process. Specifically, the inventors obtained samples of NRPCs from three different stages of the NRPC production process (i.e., from the master cell bank (MCB), working cell bank (WCB), and product stage), and co-cultured the obtained samples with DRG neurons to observe the expression of proteins related to myelination and increased neuronal stability. The inventors also obtained a sample of MSCs (from passage 14) as a control. The rate of protein expression was indicated by the intensity of fluorescence measured via a fluorescent marker on the NRPC samples. ​ is a set of immunofluorescence images showing the increased expression of nerve regeneration-related proteins of NRPCs obtained from three stages of the NRPC production process according to one exemplary embodiment of the present disclosure. As shown in ​ the stages of the NRPC production process from which samples of NRPCs were obtained are a passage 14 T-MSC culture (T-MSC P14) used as a control, a T-NRPC sample obtained from the master cell bank (MCB), a T-NRPC sample obtained from the working cell bank (WCB), and a product. As used herein, the master cell bank of T-NRPCs includes an aliquot of a single pool of T-NRPC cells differentiated from T-MSCs under defined conditions, distributed into multiple containers, and stored under defined conditions. The MCB is used to derive the working cell bank (WCB) of T-NRPCs. Thus, T-NRPCs from the WCB are at a later stage in the production process compared to T-NRPCs from the MCB. The product contains T-NRPCs prepared for treating damaged cells and derived from the WCB, thus the T-NRPCs of the product are made at a later stage in the production process compared to T-NRPCs from the WCB. After co-culturing the T-NRPC samples from each stage with DRG neurons and measuring the expression of various proteins by immunofluorescence, the inventors found that the expression of MBP and TuJl increased at each stage of the T-NRPC production process (i.e., the rate of expression increased from T-MSC P14 to MCB, from MCB to WCB, and from WCB to product. ​ The‘Nuclei’ column of illustrates the results of nuclei staining of DRG neurons co-cultured with T-NRPC samples from each stage of the NRPC production process. As shown by the nuclei stained in the‘Nuclei’ column, the number of nuclei of DRG neurons changed at each stage of the NRPC production process. Figure 5The 'Merge' column shows the merged images of MBP and TuJl protein expression and nuclear localization. By analyzing the information from the 'Nucleus' and 'Merge' columns, one can gain insight into the movement of cells involved in myelination. The increase in MBP and TuJl protein expression allows for increased cell communication via myelination and neural tube formation to be induced.

[0105] A neurite outgrowth assay involving neuroblastoma cells can be used to determine the efficacy of NRPCs in inducing neurite growth The neurite outgrowth assay demonstrates that T-NRPCs are more successful than other NRPCs in inducing neurite growth Neurites (or neuronal processes) project from the cell body of a neuron and are known to be involved in the transport of substances necessary for axon growth and regeneration, neurotransmitters, nerve growth factors, and the like. Neurite outgrowth assays can be performed to compare neurite growth induced by neuronal regeneration promoting cells, such as those described in the present disclosure. Certain clonal lines of mouse neuroblastoma cells, such as N1E-115, are known to extend or retract axons depending on the culture medium, and thus can be used in neurite outgrowth assays to test the ability of the presently disclosed NRPCs to induce neurite growth.

[0106] Figure 6A To test the efficacy of various NRPCs in inducing neurite growth, N1E-115 cells (mouse neuroblastoma cells, ATCC, USA) were cultured and seeded on microporous filters (Neurite Outgrowth Assay Kit, Millipore, USA). The seeded cells were cultured in culture medium for 48 hours, from which the culture medium was collected NRPC and MSC samples from different regions of the human body. The culture medium of each NRPC or MSC sample reflects the active ingredients (e.g., proteins) expressed and / or produced by the corresponding NRPC or MSC sample. After staining the neurites that project through the microporous filters, the absorbance was measured. It was confirmed from the neurite outgrowth assay that the culture of NRPCs modulates or stimulates the growth of neurites (axons) in N1E-115 (mouse neuroblastoma) cells. However, the inventors found that different samples of NRPCs and MSCs differentially stimulate neurite growth. Figure 6B and There are differences in the ability to induce neurite outgrowth even among tonsil-derived NRPC and MSC samplesA set of images 610 of the resulting cultures and graphs 620 and 630 quantifying neurite outgrowth are used to show the different levels of neurite outgrowth in MSCs and NRPCs derived from different regions. The different samples showing the results are AD-MSC, T-MSC, AD-NRPC, T-NRPC, negative control, positive control and primary Schwann cell groups. Graph 620 shows the average number of neurites in the cells of each sample group, while graph 630 shows the length (in pm) of the longest neurite of each sample group. The inventors found that the number of neurites and the length of the longest neurite were significantly increased in the neurite outgrowth assay group using the medium of T-NRPC samples, compared to the rest of the neurite outgrowth assay groups using the medium of the rest of the samples (T-MSC, AD-MSC and AD-NRPC samples). Thus, it was confirmed that the development of neural cells, as evidenced by neurite outgrowth on N1E-115 neuroblastoma cells caused by the medium of T-NRPC samples, can be greatly enhanced by T-NRPC.

[0107] Figure 7 A neurite outgrowth assay was also performed using the medium from different samples of MSCs from tonsils (T-MSC) and NRPCs from tonsils (T-NRPC). The different samples correspond to MSCs obtained from different people (indicated by the identification of the person in the sample name (e.g. given by numbers such as 2001, 2005, etc.)) and / or from different sides of the tonsils (indicated by "L" for left tonsil and by "R" for right tonsil in the sample name). To test the efficacy of the medium of these different T-NRPC and T-MSC samples in inducing neurite outgrowth, N1E-115 cells (mouse neuroblastoma cells, ATCC, USA) were cultured and seeded on microwell filters (Neurite Outgrowth Assay Kit, Millipore, USA). The seeded cells were cultured in the medium for 48 hours, in which the different T-NRPC and T-MSC samples were derived from different people or tonsil sides. After staining the neurites that protruded through the microwell filters, the absorbance was measured. The results of this experiment are exemplified in Figure 8A , Figure 8B and Figure 7 . In particular, Figure 8A is a set of images showing different neurite outgrowth in samples of MSCs from tonsils (T-MSC) and NRPCs from tonsils (T-NRPC). Figure 8B and Figure 7A set of graphs showing the average number of neurites and the average length of the longest neurite in the neurite outgrowth assay set based on the media of different samples corresponding to tonsil-derived MSCs (T-MSCs) and tonsil-derived NRPCs (T-NRPCs) to quantify neurite outgrowth. Further, each T-NRPC sample can correspond to a respective T-MSC sample from which it was derived, each pair of samples (T-NRPC sample and its respective T-MSC sample) corresponding to an individual human (which can be identified by a number such as 2001, 2005, 2009, etc.) and a tonsil side (which can be indicated by ‘L’ for left or ‘R’ for right in the sample name). Figure 8A , Figure 8B and Figure 7 The results shown in FIGS. 1 1-14 demonstrate that neurite outgrowth in N1E 115 cells induced by the media of the samples generally increased as the T-MSC samples differentiated into their respective T-NRPC samples. Thus, the media of T-NRPC 2001L induced more neurite outgrowth than the media of T-MSC 2001L; the media of T-NRPC 2001R induced more neurite outgrowth than the media of T-MSC 2001R; the media of T-NRPC 2009L induced more neurite outgrowth than the media of T-MSC 2009L; and the media of T-NRPC 2009R induced more neurite outgrowth than the media of T-MSC 2009R. Neurite outgrowth increased as measured by the increase in the average number of neurites in the induced N1E 115 cell samples and by the length of the longest neurite in the induced N1E 115 cell samples. However, there were outliers for T-MSC 2005R and T-NRPC 2005R. Based on the results of the neurite outgrowth assays shown in FIGS. 1 1-14, the inventors found that, while T-NRPCs generally have the ability to affect neural regeneration (e.g., by neurite outgrowth), it is critical to further screen T-NRPC samples further (e.g., based on the human and tonsil side from which the T-NRPC was derived via its respective T-MSC) to select and expand the most effective T-NRPCs. Figure 8A , Figure 8B and NRPCs can be further selected based on their ability to induce neurite outgrowth, which is determined by meeting a threshold for the average number of neurites formed The results shown in FIGS. 1 1-14 demonstrate that neurite outgrowth in N1E 115 cells induced by the media of the samples generally increased as the T-MSC samples differentiated into their respective T-NRPC samples. Thus, the media of T-NRPC 2001L induced more neurite outgrowth than the media of T-MSC 2001L; the media of T-NRPC 2001R induced more neurite outgrowth than the media of T-MSC 2001R; the media of T-NRPC 2009L induced more neurite outgrowth than the media of T-MSC 2009L; and the media of T-NRPC 2009R induced more neurite outgrowth than the media of T-MSC 2009R. Neurite outgrowth increased as measured by the increase in the average number of neurites in the induced N1E 115 cell samples and by the length of the longest neurite in the induced N1E 115 cell samples. However, there were outliers for T-MSC 2005R and T-NRPC 2005R. Based on the results of the neurite outgrowth assays shown in FIGS. 1 1-14, the inventors found that, while T-NRPCs generally have the ability to affect neural regeneration (e.g., by neurite outgrowth), it is critical to further screen T-NRPC samples further (e.g., based on the human and tonsil side from which the T-NRPC was derived via its respective T-MSC) to select and expand the most effective T-NRPCs.

[0108] NRPCs can be further selected based on their ability to induce neurite outgrowth, which is determined by meeting a threshold for the average length of the longest neurite formed Neurite outgrowth is reduced in samples involving tonsil-derived MSCs and NRPCs when expression of CD121a is reduced In particular, the inventors have discovered that NRPCs can be screened, selected, and expanded based on their ability to induce neurite growth (e.g., in neuroblastoma cells), and criteria can be established for evaluating which NRPCs most effectively induce neurite growth. In one embodiment, a given NRPC is considered capable of inducing neurite growth if the average number of neurites formed per neuroblastoma cell in a sample of corresponding neuroblastoma cells is at least 15, and the longest neurite formed in a sample of corresponding neuroblastoma cells is at least 145 µm in length. However, in some embodiments, the threshold for the number of neurites formed per neuroblastoma cell to determine the ability of an NRPC sample to induce neurite growth can be higher. For example, the threshold for the number of neurites formed per neuroblastoma cell can be approximately 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, or 100. In the implementation, the threshold can be within a range formed by selecting any two numbers listed in the preceding sentence (e.g., the minimum threshold number of neurites formed per neuroblastoma cell could be about 12, about 10 to about 30, etc.). In some aspects, the average number of neurites formed per sample can be determined by averaging the number of neurites formed in multiple samples of neuroblastoma cells. Each sample can be contained in a corresponding well. For example, the number of neuroblastoma cells (e.g., N1E-115 mouse neuroblastoma cells) used in the neurite growth assay can be 1 x 102 6 Cells / ml or 1x10 5 (Cells / well). In some aspects, multiple samples (wells) can be used. In one embodiment, a sample (well) of 20-40 N1E-115 mouse neuroblastoma cells can be used, for example, 24 samples can be used via a plate containing 24 wells. The ability of NRPC to induce neurite growth can be evaluated by measuring the number of neurites formed by each neuroblastoma cell in each sample to obtain an average value.

[0109] Figure 9A Figure 9B As previously discussed, NRPCs can be screened, selected, and expanded based on their ability to induce neurite growth (e.g., in neuroblastoma cells), and criteria can be established to assess which NRPCs most effectively induce neurite growth. In one embodiment, a given NRPC sample is considered capable of inducing neurite growth if the average length of the longest neurite formed in the corresponding neuroblastoma cell sample is at least 145 µm. In some embodiments, the threshold for the length of the longest neurite formed in the neuroblastoma cell sample (used to determine the ability of an NRPC sample to induce neurite growth in the neuroblastoma cell sample) can be higher. For example, the threshold for the length of the longest neurite formed in a sample of neuroblastoma cells can 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 embodiments, the threshold can be within a range formed by selecting any two numbers listed in the preceding sentence (e.g., the minimum threshold for the average length of the longest neurite formed in a sample of neuroblastoma cells can be approximately 150 µm, approximately 150 µm to approximately 170 µm, etc.). In some aspects, the average length of the longest neurite formed can be determined by averaging the longest lengths of neurites formed in multiple samples of neuroblastoma cells. In some aspects, multiple samples can be used (e.g., a sample of 20-40 neuroblastoma cells can be used). For example, 24 samples (enclosed in the corresponding wells) can be used via a 24-well plate. Furthermore, each well can be inoculated with 1 x 1024 samples. 5 N1E 115 mouse neuroblastoma cells. The ability of NRPC to induce neurite growth in each sample (well) can be assessed (e.g., by measuring the length of the longest neurite formed).

[0110] Figure 9A Figure 9B As previously discussed, neurite outgrowth in precursor or damaged nerve cells (e.g., mouse neuroblastoma N1E 115 cell line) can occur more effectively under certain media (e.g., those in which T-NRPCs are cultured) than other media based on active ingredients of these media. Such active ingredients can be proteins expressed by NRPCs grown in the media that have an influence on inducing neurite outgrowth. After conducting tests showing that neurite outgrowth decreases when CD121a expression is reduced, the inventors found that CD121a is a particularly important protein for neurite outgrowth. In these tests, small interfering RNA (siRNA) was used to reduce the expression of CD-121a. Specifically, the tests involved six sample groups - a negative control group, a positive control group, a sample of primary Schwann cells, a sample of T-MSCs, a sample of T-NRPCs treated with a scrambled version of siRNA, and a sample of T-NRPCs treated with siRNA. The scrambled siRNA was used as a negative control to distinguish specific gene effects from non-specific or cell-related effects. In addition, the scrambled siRNA was used to reduce non-specific effects and background noise, thereby increasing the accuracy and reliability of the experiments. CD121a expression varies among different samples of MSCs and NRPCs and among different production stages of NRPCs and Figure 10A A set of images 910 showing the results of these sample groups, and graphs 920A and 920B showing the number of neurites formed and the length of the longest neurite formed in the sample groups. As shown in Figure 10B and Figure 10A When the expression of CD121a was reduced using small interfering RNA (siRNA), neurite growth was significantly reduced in samples of T-MSCs and T-NRPCs. On the other hand, the scrambled siRNA acted to exclude the effects of siRNA transfection on the cells when knocking out the target gene. Thus, in the sample group including T-NRPCs treated with the scrambled siRNA, the number of neurites was observed to be greater than in the other experimental groups, and the length of the neurites was greater than the length of the neurites of the positive control. Thus, the inventors found that the CD121a marker that is most highly expressed in T-NRPCs is an important marker for nerve regeneration, and that when CD121a expression is reduced due to the knockdown of CD121a, the number of neurites and the length of the longest neurite decrease.

[0111] Figure 10B In light of the determination that CD121a is critical for efficient neurite outgrowth, the inventors investigated how CD121a expression varies in different kinds of MSCs and NRPCs, such as MSCs and NRPCs derived from different people, different regions of a person (including tonsil sides), or obtained from different stages of the NRPC production process. The inventors obtained several sample groups from the working cell bank, including various TMSCs, MSCs, primary Schwann cells, and controls, to test expression of CD121a - TMSC 2001L, NRPC 2001L, TMSC 2001R, NRPC 2001R, TMSC 2009L, NRPC 2009L, 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., above 75%) at the product stage. The inventors obtained these selected T-NRPC samples from the product stage of the production process to investigate how CD121a expression increased through the production process. These selected T-NRPC samples - T-NRPC 2001L, T-NRPC 2001R, T-NRPC 2009L, and T-NRPC 2009R - correspond to T-NRPCs derived from different people (identified by the number in the sample name, such as 2001, 2005, 2009, etc.) and / or different tonsil sides (as indicated by ‘L’ for left or ‘R’ for right in the sample name). The results related to CD121a expression levels are shown in FIGS. 1010, 1020, 1030, and 1040 of The quality of NRPCs is significantly improved by screening and selecting NRPC candidates with a CD121a expression level of at least 30% and Figure 10A Specifically, Figure 10B FIG. 1010 of Frozen vs. unfrozen state shows expression levels of marker CD121a in samples of MSCs and NRPCs derived from different regions and people and obtained from the working cell bank, while FIG. 1020 of

[0112] shows expression levels of CD121a in the (final) product of the sample groups of T-NRPC 2001L, T-NRPC 2001R, T-NRPC 2009L, and T-NRPC 2009R. Thawing process of cells Based on CD121a expression levels of T-NRPCs are consistently high in passages at the product stage and Figure 11ABased on the CD121a expression levels shown, the inventors have found that by screening NRPC candidates with high levels of CD121a expression, more effective NRPCs (e.g., based on the potential for improved neurite outgrowth) can be generated. In particular, the inventors have noted that, at an early stage in the NRPC production process (e.g., when MSCs are differentiated into their respective NRPCs based on GMP standards), NRPC candidates can be selected in the working cell bank (WCB) that express CD121a at expression rates exceeding 30%. For example, as shown in chart 1010, NRPC candidates from the working cell bank (WCB) that have CD121a expression levels equal to or higher than a first expression rate threshold (i.e., 30%) - T-NRPC 2001L, T-NRPC 2001R, T-NRPC 2009L, and T-NRPC 2009R - end up being T-NRPC samples that are able to express CD121a at levels higher than a higher second threshold expression rate (e.g., 80%) when these samples are at their product stage. Moreover, these samples are able to maintain sufficient expression rates (at least 30%) at least when the product stage is in a frozen state. When these samples are at their product stage, the expression rates return to expression levels exceeding 80%, at which product stage these samples are in an unfrozen state (e.g., one or more passages after thawing) and / or meet the criteria for viability. Thus, in the working cell bank (WCB) stage of NRPC candidate samples, a CD121a expression level of 30% or higher can be used as a criterion for determining which of these NRPC candidates will be selected for the remainder of the production process to form an NRPC product. However, in some embodiments, the threshold for CD121a expression levels at the working cell bank stage (e.g., the first threshold) or the product stage (e.g., the second threshold) can be higher. For example, starting from the working cell bank stage, NRPC candidates can be selected if their CD121a expression levels are at least higher than a threshold of about 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 can be within a range formed by selecting any two numbers (two percentages) listed in the preceding sentence (e.g., NRPC candidate samples can be selected if their CD121a expression rates are at least higher than a threshold of about 30%, about 30% to about 80%, about 35% to about 50%, etc.).

[0113] Figure 11B As discussed above, expression levels of CD121a appear to increase significantly when measured in one or more passages after thawing NRPCs and / or NRPC candidates from a frozen state, as compared to when in the frozen state. A frozen state can refer to NRPCs and / or NRPC candidates being cryopreserved in a suitable medium (e.g., liquid nitrogen) at a temperature below a threshold value of about -200 °C, -195 °C, -190 °C, -185 °C, -180 °C, -175 °C, -170 °C, -165 °C, or -160 °C. In embodiments, the threshold value can be within a range formed by selecting any two numbers (temperatures) listed in the preceding sentence (e.g., if NRPCs and / or NRPC candidates are cryopreserved in a suitable medium at a temperature below a threshold value, where the threshold value is between about -180 °C, about -190 °C to about -170 °C, about -180 °C to about -160 °C, etc., they can be in a frozen state). Expression rates or levels of a protein marker in NRPCs or NRPC candidates in a frozen state can be determined by measuring the expression rate or level immediately after thawing the NRPC or NRPC candidate. By measuring the expression level immediately after thawing, heat-dependent cellular activities (e.g., enzyme activities) that can affect the expression rate can be avoided. For example, a post-thaw time that is considered immediately after thawing can be less than about 0.5 seconds, 1 second, 5 seconds, 30 seconds, 1 minute, 5 minutes, 10 minutes, 30 minutes, or 1 hour. In embodiments, a post-thaw time that forms an upper limit for consideration immediately after thawing can be within a range formed by selecting any two numbers (two times) listed in the preceding sentence (e.g., a post-thaw time that forms an upper limit for consideration immediately after thawing can be within 1 minute, about 30 seconds to about 30 minutes, within 1 hour, etc.). In at least one embodiment, a cell characteristic immediately after thawing (e.g., expression level of a given marker protein in a NRPC or NRPC candidate immediately after thawing) can refer to the cell characteristic before any subsequent passage(s) (e.g., subculturing) after thawing. NRPCs and / or NRPC candidates can be said to be in a “live” state when in one or more passages after thawing from a previous frozen state. Unless otherwise specified, cells in the master cell bank (MCB) and working cell bank (WCB) stages of an NRPC production process are expected to be in a frozen state. In addition, unless otherwise specified, cells from the product (F / P) stage (e.g., T-NRPCs) are expected to be in a live state.

[0114] Figure 11A As discussed previously, to measure a characteristic from cells in a frozen state (e.g., in their working cell bank phase), the measurement can need to be performed immediately after thawing (e.g., prior to any subsequent passage(s) after thawing). Thus, measurement of a characteristic of cells in a frozen state can include thawing the cells. Cells in a frozen state (e.g., NRPCs or NRPC candidates in their working cell bank phase) can be thawed by placing the cells in an environment having a predetermined thawing temperature for a predetermined thawing period. The thawing temperature can be about 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, the thawing temperature can be within a range formed by selecting any two numbers (two temperatures) listed in the preceding sentence (e.g., the thawing temperature can be about 36°C to 38°C, about 30°C to 45°C, etc.). The thawing period can be about 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 can be within a range formed by selecting any two numbers (two times) listed in the preceding sentence (e.g., the thawing period can be about 2.5 minutes to about 3.5 minutes, about 3 minutes to about 4 minutes, etc.). In at least one embodiment, the thawing temperature is 37°C and the thawing period is 3 minutes.

[0115] Figure 11B In cases where the product of the appropriately selected NRPC (e.g., T-NRPC) was determined to express CD121a to a significant level (e.g., at least 80%), the inventors investigated how the expression level of CD121a was maintained through various passages in the product phase of the NRPC production process. The inventors obtained a subsample of T-MSCs and NRPCs at each passage from passage 5 to passage 19 and tested the subsamples for CD121a expression. CD121a expression levels correlate with neurite outgrowth in final passages of T-MSCs and T-NRPCs and Figure 12 The expression levels of CD121a for T-MSCs and NRPCs through passages up to the product are shown in Table 1110, the average expression levels for each of T-MSCs and T-NRPCs from passage 5 to passage 19 are shown in Table 1120, and a graph illustrating the expression levels of CD121a for T-MSCs and NRPCs through passages up to the product is shown. As shown in Table 1110 and Figure 1, the high CD121a expression characteristic (e.g., 80% or higher in a viable state) of T-NRPCs was maintained through passages up to the product. Figure 12 and Figures 14A-14C As shown in Table 1110 and Figure 1, the high CD121a expression characteristic (e.g., 80% or higher in a viable state) of T-NRPCs was maintained through passages up to the product. However, the expression of CD121a in T-MSC samples varied with passage and was generally lower.

[0116] T-NRPCs obtained in passages at the product stage are able to induce high neurite outgrowth The inventors also observed that in neurite outgrowth assays prepared using samples of T-NRPC products obtained in passage, the consistent high CD121a expression level of T-NRPC products in passage was consistent with consistent high neurite outgrowth. The inventors also observed that in neurite outgrowth assays prepared using samples of T-MSC products obtained in passage, the changing CD121a expression level of T-MSC products in passage was consistent with changing neurite outgrowth. Results are shown in Figure 13A Specifically, Figure 13B includes a set of images 1210 showing results of neurite outgrowth using samples up to T-NRPC and T-MSC products obtained in passage P14 to P19. In some embodiments, there is a correlation between the expression level of marker CD121a of T-MSC and NRPC at each passage up to the product, as will be described with respect to T-NRPC products are even more effective in expressing CD121a after thawing Further, the inventors assessed the state of the cells by determining cell viability and cell size.

[0117] Figures 14A-14C As previously discussed, T-NRPC samples in the product stage are able to express CD121a at a consistently high level, which correlates with high neurite growth. The inventors have demonstrated that using neurite outgrowth assays involving T-MSC and T-NRPC obtained in passage in the product stage, high neurite outgrowth is maintained. For example, Methods for generating the improved NRPCs disclosed herein include the ability of MSCs to differentiate into NRPCs and the ability of NRPCs to express CD121a and Figure 15AIndicates neurite outgrowth observed in neurite outgrowth assays involving product samples of T-MSCs and T-NRPCs obtained in passages. Here, neurite outgrowth is indicated based on the number of axons and the length of the longest axon in each sample. Specifically, chart 1310 represents the maximum length of neurites, and chart 1320 represents the average number of neurites observed in samples based on T-MSCs obtained in passages. Chart 1330 indicates the maximum length of neurites, and chart 1340 indicates the average number of neurites observed in samples based on T-NRPCs obtained in passages at the product stage (e.g., after MSCs have fully differentiated into T-NRPCs). For samples based on T-MSCs obtained in passages, the number of axons and the length of the longest axon appear to vary. However, for samples based on T-MSCs obtained in passages at the product stage, the length of the longest axon and the number of axons remain above threshold values even up to passage 19 after NRPC differentiation. Specifically, as shown in chart 1330, the length of the longest axon in samples based on T-NRPCs products appears to remain above a lower threshold of 150 pm even up to passage 19. Some samples based on T-NRPCs products even exceed a higher threshold of 300 pm. Further, as shown in chart 1340, the average number of axons in samples based on T-NRPCs products appears to remain above a lower threshold of 10 even up to passage 19. Some samples based on T-NRPCs products even exceed a higher threshold of 20.

[0118] Figure 15B As previously discussed, the inventors found that CD121a is critical for significantly improving neural regeneration, and it is known that T-NRPCs express CD121a protein particularly well at the product stage. The inventors also found that CD121a expression levels significantly increase at the product stage when compared to earlier stages (e.g., working cell bank stage). The rate of CD121a expression was measured for products of T-NRPCs after thawing and subculturing of product samples (e.g., after multiple passages). Specifically, in this study, the inventors measured CD121a expression on samples of products of T-NRPCs obtained in passages 15 to 19 after the samples were thawed (from frozen state). For comparison, corresponding T-MSC samples (in which differentiation to T-NRPCs has occurred) were also obtained and tested for CD121a expression. For consistency, the T-NRPC and T-MSC samples belonged to the human and tonsil side corresponding to 2009R (i.e., the samples were T-NRPC 2009R and T-MSC 2009R). Furthermore, to ensure that T-MSCs and T-NRPCs are viable for treatment after thawing, the inventors also tested the cell characteristics (e.g., cell viability, cell size, and cell population doubling level (cPDL)) of the samples obtained after thawing. Selecting NRPC candidates that express CD121a above a threshold level is key to generating effective NRPC products The results of these tests are shown. Specifically, graphs 1410, 1420, and 1430 show cell viability, cell size, and cell population doubling level (cPDL), respectively, of product samples of T-NRPC 2009R obtained in passages P15 to P19 compared to their corresponding T-MSC 2009R samples. Furthermore, graph 1440 shows expression of marker CD121a in product samples of T-NRPC 2009R in passages 15 to 19 compared to their corresponding T-MSC 2009R samples. As shown in these graphs, the T-MSC and T-NRPC cell samples maintained healthy basic cell characteristics through each passage. Furthermore, the T-NRPC samples maintained high CD121a expression after thawing. Specifically, a 70% expression level was observed immediately after thawing (which indicates the likely expression level of the product in frozen state), but from passage 16 onwards, the expression level recovered to over 80% (indicating the expression level in a live, non-frozen state). As previously discussed, the expression level of CD121a in T-MSC samples remained low.

[0119] Figure 10A Figure 10B Accordingly, the present disclosure describes improved NRPCs for the treatment and / or regeneration of damaged neurons and methods of generating them. As described by the foregoing experiments and tests, various embodiments of the methods of generating improved NRPCs include using MSCs obtained from human tonsils that differentiate into NRPCs expressing protein markers CD26, CD106, CD112, CD121a, and CD141, selectively expanding such T-NRPC candidates that express the protein marker CD121a above a threshold level (e.g., above 30%), and selectively expanding T-NRPC candidates that exhibit neurite outgrowth. For such embodiments, the ability of MSCs to differentiate into NRPCs is important. Figure 16A A set of images 1502-1532 is shown that illustrates the differentiation of MSCs derived from different regions into NRPCs according to one exemplary embodiment of the present disclosure. The set of images shows that MSCs form neural spheres or clusters of free-floating neural stem cells during differentiation into NRPCs. The set of images 1502-1532 demonstrates that some MSCs are better able to differentiate into NRPCs. For example, AD-MSCs and T-MSCs are well differentiated into NRPCs, while BM-MSCs and UC-MSCs are not well differentiated into NRPCs. Moreover, as previously discussed, the ability of MSCs and NRPCs to express the key CD121a protein also varies. Figure 16B A set of graphs 1540 and 1550 is shown that illustrates the expression rate of the marker CD121a in NRPCs derived from different regions of a human and their corresponding MSCs. The difference in expression levels of CD121a demonstrates that while AD-MSCs and T-MSCs are better able to successfully differentiate into NRPCs, AD-NRPCs are ultimately unable to express CD121a, while T-NRPCs are able to express CD121a at significantly higher levels (e.g., 90%). Accordingly, T-NRPCs are able to express CD121a among various NRPCs to improve the treatment of damaged neuronal cells.

[0120] Testing the improved NRPCs in mice prepared to simulate severe limb ischemia (CLI) As previously discussed with respect to Experiments to test the effects of the improved NRPCs include several administration groups and control groups and Results of experiments on CLI animal models show that T-NRPCs are more effective in improving blood flow volume compared to other treatments also as Figures 17A-17Cexpressed CD121a at a first expression level threshold of at least 30%. Furthermore, when these samples were in their product stage, these selected T-NRPC samples from the working cell bank stage were ultimately able to express CD121a at a second expression level threshold or higher (e.g., 80%). However, some samples expressing CD121a at a level below the threshold were not selected. Thus, in some embodiments, a CD121a expression level of 30% or higher in an NRPC sample at the working cell bank (WCB) stage can be used as a standard (referred to herein as a“first threshold,”“first expression level,”“first expression rate,” or“first expression level threshold”) for determining which of these NRPC samples (referred to herein as NRPC candidates) will be selected for the remainder of the production process to form a product of NRPCs. However, in some embodiments, the first threshold of CD121a expression level can be higher. For example, NRPC candidates can be selected if their CD121a expression level is at least higher than a threshold of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80%. In embodiments, the threshold can be within a range formed by any two numbers (two percentages) listed in the preceding sentence (e.g., NRPC candidate samples can be selected if their CD121a expression rate is at least higher than a threshold of about 30%, about 30% to about 40%, about 40% to about 60%, etc.). In some embodiments, samples of product NRPCs expressing CD121a higher than a second expression level threshold (e.g., 80% or higher) can be further screened. For example, in some embodiments, a method for generating effective NRPCs can include selecting NRPC candidates that meet a first threshold of CD121a expression (e.g., 30% or higher) from a working cell bank, and then selecting those NRPCs from the product stage of NRPCs that express CD121a higher than a second threshold (e.g., 80% or higher after thawing or placed in a living body standard).Even at the product stage, the inventors found that the CD121a expression rate was even further increased (e.g., to 90%) when the T-NRPC sample was thawed and subcultured one or more times. Thus, at the product stage, the T-NRPCs have an expression rate of about 90%. Figure 17A The chart 1620 side-by-side shows the expression rate of NRPC candidates selected from the working cell bank stage and the expression rate of NRPC selected from the product.

[0121] Figure 17B As will be discussed herein, the improved NRPCs described herein were tested in mice that were simulated to have critical limb ischemia (CLI) and produced favorable results. CLI is a debilitating disease in which fat deposits form on the walls of leg arteries, leading to atherosclerosis and reduced blood flow. As a result, inflammation and necrosis can occur in the tissues of the leg region. In severe cases, about 40% of patients undergo amputation, and about 20% of patients die within 6 months. To test the efficacy of the improved NRPCs disclosed herein, mice were prepared to simulate patients having CLI. Specifically, the femoral arteries of the mice’s leg sides were ligated and excised to prepare an animal model having the same pathologies as patients having CLI.

[0122] Figure 17C For the experiment, seven groups of mice were prepared to simulate CLI, with six of the groups receiving different treatments (the six groups are referred to herein as administration groups). One of the seven groups that were prepared to simulate CLI did not receive any treatment and is referred to herein as a negative control group. In addition, one group of mice was normal (i.e., not prepared to simulate CLI) and is referred to as a normal group. Clopidogrel (CP), a drug used to treat CLI, was selected to be administered in one of the six administration groups for the experiment. CP was administered in five portions to the leg muscles of the mice that had the blood vessels removed at a dose of 0.025 mg / 20 g. Four of the six administration groups of mice were administered MSCs and NRPCs. Specifically, one administration group received T-MSCs, another administration group received AD-MSCs, another administration group received T-NRPCs, and another administration group received AD-NRPCs. The NRPCs administered in two of the administration groups were different than their respective MSCs administered in the two administration groups. For the remaining administration groups, the mice received excipient (CS10). For the administration groups that received MSCs and NRPCs, a large number (e.g., 1 x 10 6 For each of the administration groups in the experiment, blood flow into the mice’s legs (measured by percent blood perfusion) was analyzed weekly for 3 weeks. For the groups of mice that were prepared to simulate CLI, the legs corresponded to the blood vessel-removed legs.

[0123] In animal models, T-NRPCs are also effective in reducing muscle fibrosis, reducing muscle inflammation, and forming capillaries Figure 18A Blood perfusion was measured in eight groups of the above experiment over a three-week period (e.g., 21 days), and pathological symptoms of leg necrosis and amputation due to inflammation were identified by visual observation. Figure 18B The results of the experiment are shown. Specifically, Figure 18A Image set 1710 shows blood flow analysis of all eight groups of animal samples over a period of time, including seven groups preparing for simulated CLI and six administration groups undergoing various forms of treatment based on MSCs, NRPCs, CS10, and CPs. As shown in this set of images 1710 illustrating blood perfusion over 21 days (D0 to D21), blood flow (shown in red) was increased in the T-NRPC administration group compared to the groups administered inducer excipient (CS10), clopidogrel (CP), T-MSCs, AD-MSCs, and AD-NRPCs. Therefore, the inventors found a reduced risk of pathological symptoms and amputation due to lower limb necrosis in the T-NRPC administration group. Results of experiments show that T-NRPCs prevent the progression of muscle fibrosis This includes a chart (1720) showing the quantification of blood flow changes over 21 days in all eight groups using a procedure specifically designed for laser Doppler imaging. Results of experiments show that T-NRPCs reduce muscle inflammation Chart 1730 includes a graph indicating the pathological conditions (e.g., necrosis, amputation, and survival) observed in the legs of mice in all eight groups on day 21 of the experiment. White bars indicate the number of intact mouse legs (e.g., no necrosis or amputation caused by the applied substance); dark gray bars indicate the number of animals with amputated mouse legs; and light gray bars indicate the number of animals with necrotic mouse limbs. As shown in Chart 1730, the groups treated with T-NRPC had a higher percentage of intact mouse legs compared to all other groups except the normal group. Therefore, animal studies demonstrate the effectiveness of the modified T-NRPC of this disclosure in treating diseases such as CLI.

[0124] Results of experiments show that T-NRPCs promote capillary formation The use of T-NRPCs in treating Charcot-Marie-Tooth disease was also studied using nerve conduction tests The ability of various treatments administered to an animal model of CLI to treat muscle fibrosis, muscle inflammation, and capillary formation over a 21-day period was also investigated. As previously discussed, the eight experimental groups from the mice in the aforementioned experiments were a normal group (i.e., mice not prepared for CLI simulation), a negative control group (i.e., mice prepared for CLI simulation and not receiving any treatment), and six administration groups of mice prepared for CLI simulation, including groups receiving CS10, CP, T-MSC, AD-MSC, AD-NRPC, and T-NRPC. PMP22 and ​A plurality of groups of images showing different degrees of muscle fibrosis, muscle inflammation, and capillary formation after the treatment period (e.g., on the 21st day after treatment in the administration groups) are shown. Specifically, group 1810 shows the treatment results for muscle fibrosis, group 1820 shows the treatment results for muscle inflammation, and group 1830 shows the treatment results for capillary formation. As shown in ​ and 18B In the group administered with T-NRPC, muscle fibrosis progressed less and showed histological findings that the shape of the muscle was maintained. The group administered with T-NRPC also showed the lowest area of inflammation and high angiogenesis. Accordingly, the inventors found that T-NRPC has therapeutic effects in a plurality of fields, such as anti-inflammatory effects, inhibition of muscle fibrosis, and neovascularization. Accordingly, the T-NRPC disclosed herein has high therapeutic applicability for severe limb ischemic diseases.

[0125] ​ To evaluate the state of muscle fibrosis in the eight experimental groups, tissues were taken from the legs of the mice on the 21st day of the experiment (e.g., after the three-week treatment period of the experiment) and analyzed for histopathology. As shown in group 1810, the image of the normal group was most similar to that of the group administered with T-NRPC, as the muscle fiber bundles in both images were distributed in an elliptical or circular shape. However, the shape of the muscle fiber bundles in the negative control group showed the general form of muscle necrosis, in which the muscle fiber bundles were not visible, and the shape of the muscle fibers was crushed. The groups treated with CS10, CP, T-MSC, and AD-MSC showed a lower level of muscle fiber recovery.

[0126] ​ To evaluate muscle inflammation in the eight experimental groups, tissues were taken from the legs of the mice on the 21st day of the experiment (e.g., after the three-week treatment period of the experiment) and analyzed for the degree of inflammatory cell infiltration by histopathology. As shown in group 1820, areas with inflammation were stained in a darker purple color, and inflammatory cell infiltration was distributed between muscle fibers or muscle blocks. In the case of the normal group, no signs of inflammation were observed. The negative control group had the most distribution of inflammatory cells. For the groups treated with CS10, CP, T-MSC, AD-MSC, and AD-NRPC, inflammation was less than in the negative control group, but more than in the group administered with T-NRPC.

[0127] ​ To assess capillary formation in the eight experimental groups, tissue was harvested from the legs of mice on day 21 after administration (e.g., after a three-week treatment period) and the extent of angiogenesis was analyzed histopathologically. As shown in group 1830, histological results showed erythrocytes clustered in rounds around the vessels, as the vessels contained endothelial cells and erythrocytes. In the normal group, the largest vessel in the mouse leg was the femoral artery. No vessels were observed in the negative control group and the CS10-treated group. However, angiogenesis was confirmed in the CP, T-MSC, AD-MSC, AD-NRPC, and T-NRPC administration groups.

[0128] ​ Peroneal muscular atrophy (CMT) is a genetic disorder affecting 1 in 2,500 people, and its phenotype and genetic causes are heterogeneous. CMT type 1A (CMT1A) is a disorder affecting peripheral nerves and is caused by peripheral myelin 22 (… ​ This study investigated the efficacy of MSCs and NRPCs (e.g., the modified T-NRPCs disclosed herein) in treating CMT1A, a hereditary neurological disorder caused by gene duplication. C22 mice were used in the experiments. C22 mice are a type of mouse with 7-8 copies of human PMP22, comprising approximately 40 kb in the proximal region, and are frequently used in studies of peroneal muscular dystrophy. Five groups of 5-week-old C22 mice were randomly selected for the experiments. Of the five groups, three received subcutaneous injections of different 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 NRPCs; another group received a moderate dose of NRPC (referred to herein as NRPC-M), containing approximately 2.5 × 10^5 NRPCs; and another group received a high dose of NRPC (referred to herein as NRPC-H), containing approximately 5 × 10^5 NRPCs. T-NRPC was administered twice to this group, with the second dose of T-NRPC given 4 weeks after the first. Another group received a percutaneous injection of CS10 (this group was referred to as "sham surgery"), while the remaining groups received no medication (this group was referred to as wild-type (W / T)).

[0129] T-NRPC administered at higher doses improves nerve conduction in mice At 16 weeks, nerve conduction tests were performed to obtain proximal responses in the calf muscles (biceps femoris), and histological analysis was performed by tissue excision. FIG. 19A to FIG. 19CResults of nerve conduction studies performed on mouse samples are illustrated by graphs 1910, 1920, and 1930. Specifically, graph 1910 shows the amplitude of the waveform associated with nerve conduction from the muscle of the lower leg (biceps femoris). Graph 1920 shows the nerve conduction velocity during nerve conduction from the muscle of the lower leg (biceps femoris). As shown in graph 1920, the nerve conduction velocity of C22 mice transplanted with a higher dose of T-NRPC (e.g., NRPC-H) was significantly increased compared to C22 mice transplanted with a lower dose of T-NRPC or the sham-operated group. Graph 1930 shows the compound muscle action potential (measured in mV) associated with nerve conduction. Again, an improvement in CMAP was observed for C22 mice transplanted with a higher dose of T-NRPC (e.g., NRPC-H) compared to C22 mice transplanted with a lower dose of T-NRPC or the sham-operated group.

[0130] T-NRPC also increases myelination of nerves in C22 mice It was also found that different levels of T-NRPC administered to the C22 mouse groups increased myelination of the nerves. FIG. 20A to FIG. 20C Three sets of images of sciatic nerves immunocytochemically stained from five C22 mouse groups from the above experiment are shown, where three of the C22 mouse groups were treated with different levels of T-NRPC (NRPC-H, NRPC-M, and NRPC-L), as described above. As predicted from the experiments performed by the inventors and previously described herein, different levels of T-NRPC resulted in different levels of protein markers available for myelination. The first set of images 2010 shows that the C22 mouse groups transplanted with different levels of T-NRPC showed an increase in expression of proteins forming myelin (e.g., MBP, shown in green) surrounding proteins that are precursors of nerve cells (e.g., NF-H, shown in red). Moreover, as the amount of T-NRPC administered to the C22 mice increased, the expression of MBP (green) and NF-H (red) increased, indicating that the formation of axons and myelin of the sciatic nerves of C22 mice can be improved with T-NRPC.

[0131] T-NRPC also improves muscle regeneration in C22 mice The experiment was also tested by detecting the expression of the marker protein MYH8 of regenerated muscle and the basement membrane protein laminin of the extracellular matrix from the immunocytochemically stained images. As shown in the second set of images 2020, the C22 mice transplanted with a higher dose of T-NRPC (e.g., NRPC-H) showed an increase in the expression of MYH8 (green) and laminin (red) compared to the C22 mice transplanted with a lower dose of T-NRPC or the sham-operated group. This indicates that the formation of muscle fibers and the extracellular matrix of the C22 mice can be improved with T-NRPC. FIG. 20BThe second set of images 2020 shows that the expression of these proteins increased in the C22 mouse group injected with T-NRPC. The shape of muscle fibers in the group treated with T-NRPC remained and returned to a level similar to that of the W / T group. The third set of images 2030 shows the expression of MYH1E and laminin, which are both skeletal muscle proteins. Again, the C22 mouse group treated with NRPC tended to have more expression of MYH1E and laminin. In addition, the shape of muscle fibers in the NRPC-H group improved to a similar degree as the W / T group.

[0132] It was also found that T-NRPC regenerated damaged nerve myelin It was also found that different levels of T-NRPC administered to the C22 mouse group regenerated the nerve myelin (e.g., where the myelin was destroyed). FIG. 21A to FIG. 21E Five sets of images are shown, which indicate the G-ratio of neurons and myelin formation of the five treated C22 mouse sample groups, including three groups treated with different levels of T-NRPC (NRPC-H, NRPC-M, and NRPC-L). In the first set of images 2110, cross sections of mouse sciatic nerves were observed with an electron microscope (TEM) to morphologically observe the myelin regeneration of axons according to the high, medium, and low concentrations of T-NRPC administered. (X3,000). In the cross sections of the nerve fibers of the sciatic nerve, round axons and myelin surrounding the axons can be observed. The second set of images 2120 shows the results after treatment. As shown in the two sets of images, the samples administered with a higher concentration of T-NRPC exhibited myelin regeneration of axons, most similar to the W / T (normal) group. For example, in the W / T group, the myelin around the axons can be normally observed. On the other hand, in the sham group, there is almost no myelin around the axons. In the groups treated with low, medium, or high concentrations of NRPC, the formation of myelin around the axons can be observed. The third set 2130 is a single image explaining the G-ratio measurement method in the nerve fiber images of the electron microscope. The fourth set of images 2140 shows that as the level of T-NRPC in the C22 mice increases, the G-ratio of the axons improves, as shown by the increasing G-ratio exhibited by the NRPC-L, NRPC-M, and NRPC-H groups, respectively, where the G-ratio exhibited by the NRPC-H group is numerically closest to the W / T (normal) group. The fifth set of images 2150 shows that in the C22 mouse groups administered with low, medium, and high concentrations of NRPC, respectively, the myelin thickness increases with increasing T-NRPC. Therefore, the experiment shows that T-NRPC increases the myelin formation of nerve cells and the myelin regeneration of destroyed myelin.

[0133] It was also found that T-NRPC expresses MPZ while modulating PMP22 to optimal levels.

[0134] As previously discussed, CMT Type 1A (CMT1A) is caused by duplication of the gene for peripheral myelin protein 22 (PMP22). While expression of PMP22 is important for myelination during development of the sciatic nerve, overexpression of PMP22 can induce CMT1A and demyelinating neuropathy. Therefore, if overexpressed, PMP22 needs to be downregulated. Meanwhile, 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 a key marker for Schwann cell development. Under healthy conditions, it is ideal for PMP22 expression to be proportional to MPZ expression. The inventors investigated whether the T-NRPC of the present disclosure has the potential to express and regulate the levels of PMP22 and MPZ in the C22 mouse groups administered with different amounts of T-NRPC (NRPC-H, NRPC-M, and NRPC-L). FIG. 22A to FIG. 22C Three sets of images and graphs indicating the expression of markers PMP22 and MPZ in C22 mouse samples are shown. 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 composite of sets 2210 and 2220, thereby showing whether the expression of MPZ and PMP22 is balanced; and the graph 2240 FIG. 22D ) indicates the expression ratio of PMP22 / MPZ for each of the C22 mouse groups.

[0135] In the W / T group, the PMP22 / MPZ ratio is 1. On the image set 2230 forming the composite of sets 2210 and 2230, the orange color of the sham group (which indicates higher PMP22 presence) contrasts with the yellow color of the W / T group and the groups administered with T-NRPC (which indicates a more balanced portion of PMP22 and MPZ). Thus, the results show that T-NRPC can effectively promote the recovery of Schwann cells and the downregulation of PMP22 by increasing the PMP22 / MPZ ratio to a value similar to the W / T (normal) group.

[0136] T-NRPC is able to modulate PMP expression by expressing miR-29a The inventors also investigated why the C22 mouse groups treated with NRPC were able to regulate the overexpression of the PMP22 gene. To confirm the mechanism of T-NRPC regulation of PMP22 expression, various microRNAs (miRNAs) of the exosomes obtained from TMSC and NRPC cultures were analyzed. FIG. 23Ais a table illustrating analysis of several miRNAs to determine which miRNAs appear to be expressed in T-MSC and T-NRPC cultures. The inventors found that the miRNA identified as miR-29a was expressed at a higher level in T-NRPC than in T-MSC cultures. MiR-29a is a microRNA that regulates PMP22 expression. Thus, the inventors found that T-NRPC are effective in treating CMT disease because T-NRPC can be used to inhibit PMP22 overexpression via expression of miR-29a. Thus, when such T-NRPC were administered to a C22 mouse model, which is the most pathogenic animal model of CMT disease characterized by overexpression of the PMP22 gene, the inventors demonstrated that PMP22 gene expression was modulated and CMT disease was improved. The inventors found that T-NRPC are characterized by increased expression of miR-29a, and that this increased expression of miR-29a allows T-NRPC to inhibit PMP22 gene expression in C22 mice, thereby treating CMT disease.

[0137] Expression of miR-29a in T-NRPC As shown in FIG. 23A and FIG. 23B , the inventors found that T-NRPC (e.g., used as a product) are characterized by increased expression of miR-29a, as evident from a fold change (fc) value of 1.329515 and a volume of 1.235058. The fold change value (fc) indicates the ratio of expression of a given microRNA (e.g., miR-29a) in T-NRPC to the expression of the microRNA in corresponding T-MSC. Thus, as shown in FIG. 23A and FIG. 23B , miR-29a is expressed 1.329515 times higher in T-NRPC than in T-MSC. In addition, the volume indicates the ratio of the expression intensity of a given microRNA (e.g., miR-29a) in T-NRPC to the expression intensity of the given microRNA (e.g., miR-29a) in corresponding T-MSC. Thus, as shown in FIG. 23A and 23BAs shown, miR-29a was expressed at 1.235058 times more strongly in T-NRPC than in T-MSC. In some embodiments, T-NRPC can be selected (e.g., for use in making a product) based on a fold change value (fc) of those expressing miR-29a (when compared to expression of miR-29a in corresponding T-MSC) that is at least above a threshold of about 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 can be within a range formed by selecting any two numbers (two fold change values (fc)) listed in the preceding sentence (e.g., T-NRPC can be selected based on a fold change value (fc) of those expressing miR-29a that is above a threshold of about 1.1 to about 1.5, about 1.15 to about 1.25, etc.). In some embodiments, T-NRPC can be selected (e.g., for use in making a product) based on a volume of miR-29a of the T-NRPC (i.e., a ratio of the expression strength of miR-29a in the T-NRPC to the expression strength of miR-29a in the corresponding T-MSC) that is at least above a 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. In some embodiments, the threshold can be within a range formed by selecting any two numbers (two volumes) listed in the preceding sentence (e.g., T-NRPC can be selected based on a volume that is above a threshold of about 1.0 to about 1.5, about 1.15 to about 1.25, etc.). FIG. 23A and FIG. 23B Further shown is the use of miR-413 as an endogenous control, which can have a constant expression rate, and can be used to quantitatively compare the amount of miR-29a expressed in T-MSC and T-NRPC. For example, as shown in FIG. 23A and FIG. 23B As shown, the expression rate of miR-29a in T-NRPC (measured by fold change value (fc) and volume) is about 4 times higher than in T-NRPC compared to the expression rate of miR-413.

[0138] The inventors also found the average (mean) cycle threshold (Ct) value for miR-29a. It is expected that the average Ct value will be faster if there is more target gene, but slower if there is less target gene. The inventors found that the average Ct value for miR-29a in T-NRPC is 18.208, which is faster than the average Ct value for miR-29a in T-MSC, and thus found that more miR-29a is present in T-NRPC.

[0139] T-NRPC is effective for treatment of any ischemic tissue While the above animal studies were performed on Balb / c nude mice that mimic CLI, it is expected that the disclosed T-NRPCs will be able to provide the disclosed beneficial effects on any ischemic tissue. It is well known that ischemia refers to a restriction in blood supply to any tissue, muscle group, or organ of the body, resulting in a lack of oxygen to the tissue. Due to ischemia, the damaged tissue can result in damaged nerves (e.g., demyelination and inadequate nerve conduction), muscle fibrosis, muscle inflammation, and reduced blood flow, which are characteristics of the nerves and tissues described in FIGS. 19-23. The beneficial effects of T-NRPC delivery to the ischemic tissue of the lower limbs of C22 mice can thus be applied to other ischemic tissues and / or other animals (e.g., humans).

[0140] Administration and formulation of the compositions described herein The pharmaceutical composition of the present disclosure can be administered orally or parenterally. Specifically, it can be administered parenterally, for example, by intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intravitreal injection, subretinal injection, suprachoroidal injection, eye drop administration, intracerebroventricular injection, intrathecal injection, intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisternal injection, intracoronary injection, intracranial injection, intradural injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection, intraperitoneal injection, intrapleural injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection, local injection, etc.

[0141] The administration dose of the pharmaceutical composition of the present disclosure can vary depending on various factors such as the formulation method, the administration method, the administration time, the administration route, the reaction and its degree reached by the administration of the pharmaceutical composition, the age, body weight, general health condition, pathological condition or severity, gender, diet and excretion rate of the subject to whom the pharmaceutical composition is administered, and other drugs or ingredients used together and similar factors well known in the medical field, and the administration dose required for the treatment effective can be easily determined and prescribed by one of ordinary skill in the art.

[0142] The administration route and the administration method of the pharmaceutical composition of the present disclosure can be independent of each other, and are not particularly limited as long as the pharmaceutical composition can reach the target site.

[0143] Formulation of the compositions described herein for administration to a subject In at least one embodiment, the formulation of the final product to be administered can be in a frozen state. In some aspects, the formulation can be thawed according to the thawing methods of the cells described above prior to administration. In other aspects, the formulation can be administered in a frozen state. Formulations for parenteral administration can include sterile 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, and the like can be used. As a base for suppositories, witepsol, polyethylene glycol, Tween 61, cocoa butter, lauric oil, glycerogelatin, and the like can be used. The pharmaceutical compositions of the present disclosure can be prepared as single-dose or multi-dose formulations. In some aspects, the pharmaceutically acceptable carriers and / or excipients can be used in the formulation according to methods readily implemented by one of ordinary skill in the art to which the present disclosure pertains. The pharmaceutical compositions according to the present disclosure can be prepared into various formulations according to conventional methods. The compositions of the present disclosure can contain one or more active ingredients known to have a preventive or therapeutic effect on neurological diseases as well as stem cell-derived neuron regeneration-promoting cells having a neuron regeneration activity.

[0144] Dose of administration The administration dose of the pharmaceutical composition of the present disclosure can vary depending on various factors such as the formulation method, the administration method, the administration time, the administration route, the reaction and its degree reached by the administration of the pharmaceutical composition, the age, body weight, general health condition, pathological condition or severity, gender, diet, and excretion rate of the subject to whom the pharmaceutical composition is administered, and other drugs or ingredients used together and similar factors well known in the medical field, and the administration dose required for the treatment effective can be easily determined and prescribed by one of ordinary skill in the art. In at least one embodiment, the administration dose can comprise the T-NRPC described herein in an amount of about 1 x 10 5 cells / kg to about 1 x 10 7 cells / kg. In another embodiment, the administration dose can comprise about 1 x 10 4 cells / kg, 5 x 10 4 cells / kg, 1 x 10 5 cells / kg, 5 x 10 5 cells / kg, 1 x 10 6 cells / kg, 5 x 10 6 cells / kg, 1 x 10 7 cells / kg, 5 x 10 7 cells / kg, or 1 x 10 8The dosage is the amount of T-NRPC described herein in cells / kg. In some embodiments, the administration dose may contain the T-NRPC described herein within a range formed by selecting any two numbers listed in the preceding sentence (e.g., the administration dose may contain about 5 × 10⁻⁶ cells / kg). 4 Cells / kg to approximately 5×10 7 cells / kg, approximately 1×10 5 One cell / kg to approximately 1×10 6 The amount of T-NRPC described herein is in the form of cells / kg, etc. In some respects, the amount of T-NRPC described herein in the administered dose may depend on a variety of factors, including the subject's age, weight and sex, the disease to be treated and its degree and severity. In some embodiments, the administered dose may contain T-NRPC as the active ingredient as described herein, comprising 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%, 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% of the volume of the administered dose. In some implementations, the threshold percentage volume can be within a range formed by selecting any two numbers (two percentage volumes) listed in the preceding sentence (e.g., the administered dose can have a threshold of at least about 0.2% to about 2%, about 1% to about 15%, about 2% to about 10%, etc., which is described as the active ingredient T-NRPC). Example Example 1.1 – Obtaining T-MSCs Left and right tonsil tissues obtained from numerous donors at Ewha Womans University College of Medicine were isolated and placed in 10 mL DPBS (Dulbecco's phosphate-buffered saline) supplemented with 20 μg / mL gentamicin. The tissues were centrifuged at 1,500 rpm for 5 minutes and then washed twice. The washed tonsil tissues were sectioned using sterile scissors.

[0145] 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 the same weight of enzymatic reaction solution. The composition of the enzymatic reaction solution is described in Table 1.

[0146] [Table 1]

[0147] 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 DPBS and then centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and the remaining pellet was resuspended in 10 mL DPBS to prepare a suspension. The suspension was passed through a 100-μm filter. The tonsil-derived mesenchymal stem cells remaining in the filter were washed with 20 mL DPBS and then centrifuged at 1,500 rpm for 5 minutes. After centrifugation, the supernatant was removed, and after adding ACK lysis buffer, incubated at 37°C in a thermostatic 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 a cell suspension. Then, the number of cells in the prepared cell suspension was counted. The cell suspension was inoculated in a T175 flask and incubated at 37°C in a 5% CO2 incubator.

[0148] Example 1.2 - Obtaining AD-MSCs The adipose-derived mesenchymal stem cells were purchased from Lonza (Human Adipose-Derived Stem Cells, Catalog No. PT-5006, Lonza, Switzerland). The adipose-derived mesenchymal stem cells were cultured using the medium (Bulletkit ADSD, Catalog No. PT-4505) provided by Lonza.

[0149] Example 1.3 Measurement of population doubling time As previously discussed, the population doubling time refers to the time required for the population size to double. After obtaining the MSC sample for culture, the inventors used a flow cytometer to determine the cell count data. Then, the inventors used the cell count data to calculate the population doubling time. Specifically, the inventors relied on the formula, Doubling Time (hours) = (t - t0) x log(2) / log(N t / N0), where t is the time at the end of the measurement period, t0is the time at the beginning of the measurement period, N tNtis the cell count at time t, and No is the initial cell count at time to.

[0150] Example 2. Formation of neurospheres Neurospheres were formed by culturing the mesenchymal stem cells of Example 1. Specifically, the mesenchymal stem cells were subcultured to 4-7 passages. After removing the culture medium, the mesenchymal stem cells were washed with DPBS. After treating the washed cells with TrypLE, the harvested cells were counted. After centrifuging the harvested cells and removing the supernatant, they were resuspended in a neurosphere formation medium. The composition of the neurosphere formation medium is described in Table 2.

[0151] [Table 2]

[0152] The cells resuspended in the neurosphere (1 x 10 6 cells) formation medium were seeded onto ultra-low attachment culture dishes (60 mm). The seeded cells were cultured for 3 days at 37°C and 5% CO2. After culturing for 3 days, the neurospheres formed on the culture dishes were collected in 15-mL test tubes. After centrifuging the collected cells and removing the supernatant, a neurosphere suspension was prepared by adding fresh neurosphere formation medium. The neurosphere suspension was transferred to an ultra-low attachment culture dish, and the neurospheres were cultured for 4 days at 37°C and 5% CO2.

[0153] Example 3. Use of neurospheres to differentiate into candidate cells for neuron regeneration promoting cells (NRPCs) The neurospheres formed in Example 2 were finely crushed using a 23-26G syringe needle. The crushed neurospheres were transferred to 15-mL tubes using a pipette, followed by centrifugation. After removing the supernatant, the crushed neurospheres were resuspended by adding neuron regeneration-promoting cell induction medium into the tubes. Various neuron regeneration-promoting cell induction media were prepared by mixing three or more of the following in DMEM / F12 containing GlutaMAX: 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 Forskolin (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), 8) 0.01-1 ng / mL sonic hedgehog (SHH, R&D Systems, USA), 9) 1-10 ng / mL PDGF-AA (platelet-derived growth factor-AA, Peprotech, USA), and 10) 50-300 ng / mL heregulin-β1, Peprotech, USA).

[0154] The neurospheres resuspended in various media were inoculated onto T175 flasks coated with laminin (2 μg / mL). The inoculated neurospheres were cultured for 8-10 days, while the neuron regeneration-promoting cell induction medium was changed every 3 days. FIG. 1 ).

[0155] Example 4. Screening of neuron regeneration-promoting cells by analyzing CD marker expression Among the neuron regeneration-promoting cell candidates from Example 4 and the neuron regeneration-promoting cells differentiated therefrom, the expression of a total of 242 CD markers was analyzed in tonsil-derived mesenchymal stem cells (T-MSCs) in which myelination was confirmed morphologically.

[0156] To analyze the CD markers, 3x10 7 Target cells were collected. The target cells were washed with DPBS, followed by centrifugation at 2000 rpm for 5 minutes. After removing the supernatant and washing once with DPBS, centrifugation was performed, and the remaining pellet was resuspended in 30 mL of FACS buffer. 100 μL of the cell suspension (1x10 5Each well of a round-bottom 96-well plate was seeded with 5,000 target cells (e.g., a cell line or primary cells). Then, 10 μL of a primary antibody for a CD marker was added to each well of the 96-well plate. After incubation for 30 minutes on ice, each well was washed with 100 μL of FACS buffer and then centrifuged at 300 g for 5 minutes. After removing the supernatant and adding 200 μL of FACS buffer to each well, centrifugation was performed 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 completed, the supernatant was removed and then 100 μL of the prepared secondary antibody was added to each well. After incubation for 20-30 minutes on ice, 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 by adding 200 μL of FACS buffer to each well, and the expression of the CD marker in the target cells was investigated by flow cytometry or FACS (fluorescence-activated cell sorting).

[0157] Results comparing the expression of CD markers in induced neuron regeneration-promoting cells are shown in FIG. 3 As shown in FIG. 3 , neuron regeneration-promoting cells (NRPCs) and mesenchymal stem cells (MSCs) showed similar CD marker expression patterns, but showed differences in the expression patterns of some markers. The CD marker expression patterns of MSCs and corresponding NRPCs differentiated from MSCs were compared to select CD markers with increased or decreased expression as differentiation markers for neuron regeneration-promoting cells. CD markers with increased or decreased expression in NRPCs are shown in FIG. 3 As shown in FIG. 3 , CD markers with increased expression in NRPCs (CD106, CD112, CD121a, CD338, etc. when compared to corresponding MSCs from which the NRPCs were derived, and such expression was most prominent for tonsil-derived NRPCs (T-NRPCs) differentiated from tonsil-derived MSCs (T-MSCs). Also, as shown in FIG. 3 , CD markers with decreased expression included CD26, CD54, CD141, etc. In addition, as shown in FIG. 3 ​As shown, CD121a expression was particularly high in T-NRPCs compared to other NRPCs. Thus, as discussed previously, the role of CD121a expression was further investigated and found to be particularly relevant to promoting neurite outgrowth. From the above results, CD markers that were generally increased or decreased in expression in T-MSC-derived tonsil-derived neuron regeneration promoting cells (T-NRPCs) were screened. The screened markers are as follows: CD markers that were generally increased in expression: CD106, CD112, and CD121a. CD markers that were generally decreased in expression: CD26 and CD141. The pattern of CD markers that were generally increased or decreased in expression was also similarly observed in neuron regeneration promoting cells differentiated from adipose-derived mesenchymal stem cells of Examples 1-2. Thus, it was found that CD markers - CD26, CD106, CD112, CD121a, and CD141 - can be used to identify NRPCs differentiated from MSCs. As discussed herein, while markers CD121a, CD106, CD112, CD26, and CD141, whose expression generally changes, can be used as differentiation markers for neuron regeneration promoting cells, the inventors found that the high expression rate of CD121a is particularly useful for identifying effective NRPCs.

[0158] Example 4. Cytokine array assay of neuron regeneration promoting cells As FIG. 4A and FIG. 4B Expression of 507 cytokines was analyzed in T-MSCs and AD-MSCs, as well as the corresponding NRPCs - T-NRPCs and AD-NRPCs - differentiated therefrom, as shown. Target cells were cultured for analysis of cytokines. Target cells were seeded in flasks and cultured for 3-4 days. When the target cells filled 80% or more of the flask area, the culture medium was removed and the target cells were washed twice with DPBS. After washing, the culture medium was replaced with DMEM (Dulbecco’s Phosphate-Buffered Saline) without FBS (fetal bovine serum), cytokines, etc., to exclude the influence of cytokines. After 30 hours of culture, the culture of target cells was collected.

[0159] 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 conditioned medium passing 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 a Bradford assay. The concentrated culture was adjusted to a final concentration of 1 mg / mL by mixing with DMEM.

[0160] The membrane coated with antibodies capable of detecting 507 cytokines (Cytokine Array Kit, RayBiotech, USA) was reacted for 30 minutes by treating with blocking buffer. After removing the blocking buffer remaining on the membrane and replacing with concentrated culture, the membrane was reacted overnight in a refrigerator. The membrane was washed with washing buffer 7 times. After adding HRP-conjugated streptavidin solution, the membrane was reacted at room temperature for 2 hours. After removing the HRP-conjugated streptavidin solution, the membrane was washed with washing buffer 7 times. After washing, the membrane was soaked with ECL (enhanced chemiluminescence) reagent, and the expression of cytokines was confirmed using an imaging device.

[0161] The results of the expression of cytokines in the neuron regeneration-promoting cells were compared using the heat map shown in FIG. 11. In FIG. 11, AD-MSC, AD-NRPC, T-MSC, and T-NRPC showed different expression patterns. As previously discussed, the expression of cytokines HGF, µPA, and GRO-α, which are particularly relevant to nerve regeneration, was higher in NRPCs than in MSCs, especially in T-NRPCs. FIG. 4A FIG. 4B The results of the expression of cytokines in the neuron regeneration-promoting cells were compared using the heat map shown in FIG. 11. In FIG. 11, AD-MSC, AD-NRPC, T-MSC, and T-NRPC showed different expression patterns. As previously discussed, the expression of cytokines HGF, µPA, and GRO-α, which are particularly relevant to nerve regeneration, was higher in NRPCs than in MSCs, especially in T-NRPCs. FIG. 4A FIG. 4B The results of the expression of cytokines in the neuron regeneration-promoting cells were compared using the heat map shown in FIG. 11. In FIG. 11, AD-MSC, AD-NRPC, T-MSC, and T-NRPC showed different expression patterns. As previously discussed, the expression of cytokines HGF, µPA, and GRO-α, which are particularly relevant to nerve regeneration, was higher in NRPCs than in MSCs, especially in T-NRPCs.

[0162] Example 5. Screening of neuron regeneration-promoting cell candidate cells by confirming myelination of peripheral nerves It was investigated whether the neuron regeneration-promoting cell candidates prepared in Example 3 had the ability to myelinate peripheral nerves. Specifically, the differentiated neuron regeneration-promoting cell candidates were co-cultured with dorsal root ganglion (DRG), and it was investigated whether myelination occurred.

[0163] Dorsal root ganglion (DRG) cells isolated from rats were purchased from Lonza (Rat Dorsal Root Ganglion Cells, Catalog No. R-DRG-505, Lonza, Switzerland). The candidate cells were co-cultured with the purchased dorsal root ganglion. The DRG cells were cultured using the medium provided by Lonza (Primary Neuron Growth Medium bullet kit (PNGM), Catalog No. CC-4461).

[0164] The medium was replaced every 3 days. Due to the co-culture of the candidate cells with the dorsal root ganglion, it was confirmed that myelination was achieved in some cells in terms of cell morphology. FIG. 5 - FIG. 8B Specifically, as previously discussed, the inventors determined that the number of axons and the length of the longest axon were significantly increased in the assay group using T-NRPCs in the medium compared to the assay groups using T-MSCs, AD-MSCs, or AD-NRPCs in the medium.

[0165] It will be apparent to those skilled in the art that various modifications and variations can be made in the exemplary implementations described herein without departing from the scope or spirit of the disclosure ​​ It should be understood that various changes and modifications to the exemplary embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the subject technology and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. Neuronal regeneration promoting cells (NRPCs) are induced from tonsil-derived mesenchymal stem cells and express CD26, CD106, CD112, CD121a and CD141, with CD121a having an expression level of approximately 75% or higher.

2. The NRPC of claim 1, wherein the NRPC is in a passaged state during one or more passages after thawing from a frozen state, wherein the expression level of CD121a is measured in the passaged state.

3. The NRPC of claim 1, wherein the expression level of CD121a in the NRPC is about 90% or higher, wherein the NRPC is in a passaged state during one or more passages after thawing from a frozen state, wherein the expression level of CD121a is measured in the passaged state.

4. The NRPC as described in claim 1, The expression level of CD26 in the NRPC was approximately 5% or lower. The expression level of CD106 in the NRPC was approximately 15% or higher. The expression level of CD112 in the NRPC was approximately 50% or higher, and The expression level of CD141 in the NRPC was approximately 30% or lower. 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 as described in claim 1, The expression level of CD26 in the NRPC is approximately 10% to approximately 35%. The expression level of CD106 in the NRPC is approximately 10% to approximately 35%. The expression level of CD112 in the NRPC is said to be from about 25% to about 90%, and The expression level of CD141 in the NRPC is approximately 10% to approximately 45%. The NRPC is in a passaged state during one or more passages after thawing from a frozen state, wherein the expression levels of CD26, CD106, CD112 and CD141 are measured in the thawed state.

6. A method for generating an NRPC as claimed in claim 1, the method comprising: Multiple tonsil-derived mesenchymal stem cell (MSC) cultures were generated to form neurospheres; Multiple cell cultures were generated from the neurosphere to induce NRPC candidates; Freeze at least a portion of the NRPC candidates; Thaw the multiple NRPC candidates from their frozen state; The expression level of CD121a was measured immediately after the plurality of NRPC candidates were thawed from their frozen state; and Among the plurality of NRPC candidates, an NRPC expressing CD26, CD106, CD112, CD121a and CD141 is selected, wherein the expression of CD121a is at or above a first expression level, wherein the first expression level is approximately 30%.

7. The method of claim 6, wherein each of the plurality of tonsil-derived MSC cultures is generated in a separate container such that each of the containers contains a separate culture comprising tonsil-derived MSCs and a culture medium for forming the neurosphere.

8. The method of claim 7, wherein the method further comprises, prior to generating the plurality of cell cultures from the neurosphere: Collect the nerve ball from each of at least a portion of the container containing the nerve ball; and The collected neurospheres are processed to further collect cells from them.

9. The method of claim 8, wherein each of the plurality of cell cultures from the neurosphere is generated in a separate container such that each of the containers contains a separate culture comprising collected cells from the neurosphere and a culture medium for inducing the cells into NRPC candidates.

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

11. The method of claim 6, wherein generating the plurality of tonsil-derived MSC cultures comprises: Provides tissue samples of a person's left and right tonsils; MSCs derived from the first tonsil were isolated from the left tonsil tissue; as well as MSCs derived from the second tonsil were isolated from the right tonsil tissue.

12. The method of claim 6, further comprising: For each or a subset of the plurality of NRPC candidates, evaluate whether the NRPC candidate induces myelination in the dorsal root ganglion. The NRPCs selected in the dorsal root ganglia are those that induce myelination and express CD26, CD106, CD112, CD121a, and CD141, wherein the expression of CD121a is at or above the first expression level.

13. The method of claim 12, wherein the evaluation comprises: Co-culture of dorsal root ganglia and the NRPC candidate to be evaluated; as well as The dorsal root ganglion was then examined and myelin sheath formation was confirmed.

14. The method of claim 6, further comprising: For each or a subset of the plurality of NRPC candidates, an evaluation is made as to whether the NRPC candidate induces neurite growth on a sample of the corresponding neuroblastoma cells, wherein a given NRPC candidate is determined to induce neurite growth when the average number of neurites formed per neuroblastoma cell in the sample of the corresponding neuroblastoma cells is at least 15, and the longest neurite formed in the sample of the corresponding neuroblastoma cells is at least 150 µm in length; and Among the NRPC candidates, the NRPC that induces the neurite ingrowth and expresses CD26, CD106, CD112, CD121a and CD141 is selected, wherein the expression of CD121a is at or above the first expression level.

15. The method of claim 6, further comprising: Amplify the selected NRPC within multiple passages; NRPC is harvested from at least one of the multiple generations; as well as NRPC harvested by freezing.

16. The method of claim 15, further comprising: After amplification within at least one of the aforementioned multiple passages and further selection of NRPCs before harvest, The NRPCs further selected are those with CD121a expression levels equal to or higher than the second expression level when measured during one or more passages after thawing. The second expression level is 75%.

17. A method for treating damaged nerve cells, the method comprising: The composition comprising NRPC as described in claim 1 is administered to a subject having said damaged nerve cells in an effective amount for inducing myelination of damaged nerve cells or myelination of Schwann cells.

18. A method for treating muscle fibrosis, the method comprising: The composition comprising NRPC as described in claim 1 is administered to a subject suffering from said muscle fibrosis in an effective amount for treating said muscle fibrosis.

19. A method for treating muscle inflammation, the method comprising: The composition comprising NRPC as described in claim 1 is administered to a subject suffering from said muscle inflammation in an effective amount for treating said muscle inflammation.

20. A method for inducing angiogenesis in ischemic tissue, the method comprising: The composition comprising the NRPC as described in claim 1 is administered to a subject with ischemic tissue in an effective amount for inducing angiogenesis.

21. A method for treating severe limb ischemia (CLI), the method comprising: The composition comprising the NRPC as described in claim 1 is injected into a subject suffering from the CLI at an effective amount for treating the CLI.

22. A method for treating peripheral nerve injury, the method comprising: The composition comprising the NRPC as described in claim 1 is administered to a subject suffering from the peripheral nerve injury in an effective amount for treating the peripheral nerve injury.

23. A method for inhibiting the overexpression of peripheral myelin 22 (PMP22), the method comprising: The composition comprising the NRPC as described in claim 1 is applied in an effective amount for inhibiting the overexpression of PMP22, at least in a local region, to the local region of the body of a subject in which the overexpression of PMP22 is confirmed or evaluated.

24. The method of claim 23, wherein the method treats the subject with peroneal muscular atrophy (CMT).

25. The method of claim 23, wherein the composition is applied immediately after the NRPC is thawed from its frozen state.

26. A method for increasing miR-29a expression, the method comprising: The composition comprising the NRPC as described in claim 1 is applied in an effective amount for increasing miR-29a expression, at least in a localized region, to the localized region of the body of a subject in which the need for increased miR-29a expression is confirmed or evaluated.

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

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