MESENCHYMAL STROMAL CELLS AS A REPROGRAMMING SOURCE FOR IPSC INDUCTION

Abstract

Provided are mesenchymal stromal cells as a reprogramming source for ipsc induction. In particular, Provided is a method for generating induced mesnchyaml stromal cells (iMSCs), the iMSC generated by the method as well as the use thereof.

Claims

1. A method for generating induced mesenchymal stromal cells (iMSCs) comprising: culturing induced pluripotent stem cells (iPSCs) under a first medium for a first predetermined period; replacing the first medium with a second medium and culturing the cells under the second medium for a second predetermined period; trypsinizing the cultured cells; seeding the trypsinized cells on a coated or non-coated tissue culture under a third medium for a third predetermined period; and replacing the third medium with a fourth medium and culturing the seeded cells for a fourth predetermined period.

2. The method of claim 1, wherein the iPSCs are generated by reprogramming human primary mesenchymal stromal cells (MSCs).

3. The method of claim 1, wherein the first medium comprises: a knockout serum; and a TGF beta and ALK inhibitor.

4. The method of claim 1, wherein the second medium comprises: a Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12); a TGF beta and ALK inhibitor; and a knockout serum or an Insulin-Transferrin-Selenium (ITS-G) solution.

5. The method of claim 1, wherein the third medium comprises: DMEM/F12; a knockout serum; and a basic fibroblast growth factor (bFGF) and an epidermal growth factor (EGF).

6. The method of claim 1, wherein the fourth medium comprises DMEM-LG medium and is optionally supplemented with FBS.

7. A method for generating induced mesenchymal stromal cells (iMSCs) comprising: generating induced pluripotent stem cells (iPSCs) by repromramming mesenchymal stromal cells (MSCs); and generating the iMSC by differentiation of the iPSCs.

8. A method for generating induced mesenchymal stromal cells (iMSCs) by differentiation of the iPSCs, wherein the iPSCs are generated by repromramming mesenchymal stromal cells (iMSCs).

9. The method of claim 7, wherein the MSCs are human primary MSCs.

10. A medium for generating or inducing induced mesenchymal stromal cells (iMSCs) comprising: a Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12); a knockout serum; and; a TGF beta and ALK inhibitor.

11. The medium of claim 10, further comprising: a cytokine; and an epidermal growth factor (EGF).

12. The medium of claim 11, further comprising: a bovine serum albumin; and an Insulin-Transferrin-Selenium (IT S-G) solution.

13. An induced mesenchymal stromal cell (iMSC) generated from the method of claim 1.

14. A composition comprising a plurality of iMSCs of claim 13 and optionally a carrier, an excipient, or a diluent.

15. A method for treating a disease in a subject, comprising administrating an effective amount of the iMSCs of claim 13 into the subject.

16. The method of claim 15, wherein the disease is selected from an autoimmune disease and an inflammatory disease.

Description

DESCRIPTION OF DRAWINGS

[0047] FIG. 1. Characterization of pluripotency of iPSCs derived from human MSCs. (A) Immunostaining with fluorescent antibodies of iPSCs specific markers of SEEA4, TRA-1-60 and TRA-1-81 as well as transcriptional factor SOX2 and with DAPI. (B) Xenografts of human iPSC cells generate well-differentiated teratoma-like masses containing three embryonic germ layers. Immunodeficient mouse recipients were injected with human iPSCs intramuscularly. Resulting teratomas demonstrated the following features in ectoderm B(1): neural rosettes, mesoderm B(2): muscle, and endoderm B(3): respiratory epithelium. (C) and (D): in vitro formation of embryoid body at day 7 in vitro. qPCR analyzed the relative expression levels of different respective genes of the three germ layers: Ectoderm (SOX1, OTX2, GBX2, PAX6); Mesoderm (TBC, GSC, MIXL); and Endoderm (SOX17, FOXA2, AFP). (E) Karyoview of iPSC_MD analyzed by SNP array.

[0048] FIG. 2. Characterization of iMSCs. (A) Cell surface marker evaluation was performed for the presence of the typical MSC markers, including CD73.sup.+, CD90.sup.+, CD105.sup.+ and CD44.sup.+ while CD14.sup.−, CD19.sup.−, CD45.sup.− and HLA-DR.sup.−. The expressions of most markers were similar, but CD44 present higher in iMSC-MD compared to the other two kinds of stem cells. (C) Comparison of the proliferation and survival during iMSCs induction and passage process. Although the proliferating status was similar during the iMSC induction, the survival and adherent percentage was much higher in iPSC-MD compared to the other two sources stem cells. With or without coating, iMSCs induced from iPSC-MD had a much higher survival rate. All experiments were triplicate. The experiments of iPSC-MD were repeated by using iPSCs derived from at least 2 donors.

[0049] FIG. 3. Comparison of the RNA expression levels among iMSCs from different sources. iMSC_MD1 and MD2 all showed more similar profile to primary MSCs. Sequencing reads are mapped back to human mRNA reference sequences, FRKM was used to present gene expression levels, and DESeq package was used for differential gene expression. Gene with an adjusted P-value <0.05 found by DESeq were assigned as differentially expressed.

[0050] FIG. 4. Comparison of the protein profiles of secreted and cell lysate between primary MSCs and iMSCs_MD1. Levels of forty different soluble human protein were detected using Human XL cytokine array. Medium was combined 3 independent iMSC conditional medium. Results showed that secretion profiles were similar between the primary MSCs and iMSCs (from the same donor). They were summarized into 3 groups: (A) and (D) increased release or expression (>1.5 fold), or (B) and (E) decreased release or expression (<0.65 fold) or (D) and (F) relatively similar on release or expression (0.65<x<1.5 fold). (G) and (H) hepatocyte growth factor (HGF) in both iMSC_MD1 and primary MSCs MD1 was evaluated by using Human HGF ELISA kit. Meanwhile, the HGF secretion level was reduced after using CD44 neutralizing antibodies. Medium was changed after incubation with antibodies about 12 hrs and replaced with DMEM-LG+2% FBS for 12 hrs before supernatant collection (n=3).

[0051] FIG. 5. HGF enriched conditional medium of iMSCs and MSCs ameliorated active EAE symptoms in treatment protocol (n=4-5) and inhibited inflammation and demyelination in the lumbar spinal cord of the EAE mice. Mice were administrated via i.g. with vehicle (n=5) or iMSC-CM treatment (n=4) or MSC-CM treatment (n=4) (3 mg/kg/day) once the other day starting from the day 18 of EAE induction, which reached the peak score (A). (B) Accumulative clinical scores of treatments for up till 30 dpi were calculated. Data presented as mean. Mean clinic scores were improved and demyelination and lymphocyte infiltration were reduced with both treatments compared to EAE mice. (C) Pathology scores of inflammation and demyelination for H7E and LFB images respectively. Lumbar spinal cords (L4-L6) from treated EAE mice with vehicle or CMs from iMSCs or MSCs were obtained at 30 dpi (3 mg/mouse/day) and stained by H&E (D) and Luxol Fast Blue (LFB) (E) (scale bar, 100 μm).

[0052] FIG. S1. Representative images of iPSC_MD cells (Passage 10) colony reprogrammed from hMSCs by Sendai viral transduction.

[0053] FIG. S2. Characterization of iPSCs specific markers and transcriptional factors by flowcytometry.

[0054] FIG. S3. Detection of clearance of Sendi virus by semi-PCR.

[0055] FIG. S4 and FIG. S5. Simple component size examination of conditional medium from iMSCs and MSCs by NanoSight NS300.

[0056] FIG. S6. Representative images of cells under different MOI of viral transfection. 1.75× MOI: KOS MOI=8.75, hc-Myc MOI=8.75, hK1f4 MOI=4.5; 2× MOI: KOS MOI=10, hc-Myc MOI=10, hKlf MOI=6).

[0057] FIG. S7. Representative images of cells under different MOI and medium change strategies. Gradient 1: Day 9 change: 50% of (DMEM-LG with 10% FBS)+50% of E8 medium; Day 10 change: 100% of E8 medium. Gradient 2: Day 9 change: 70% of (DMEM-LG with 10% FBS)+30% of E8 medium; Day 10 change: 30% of (DMEM-LG with 10% FBS)+70% of E8 medium; Day 11 change: 100% of E8 medium.

EXAMPLES

[0058] The following examples serve to provide further appreciation of the invention but are not meant by any way to restrict the effective scope of the invention.

Example 1. Reprogramming Human MSCs into iPSCs

[0059] To compare characteristic of iMSCs derived from ES cells or iPSCs of different origins, one human ES cell line (H7) and two iPSC cell lines were included. The two iPSC cell lines are derived from human fibroblast (iPSC_Fb) and human primary bone marrow derived MSCs (iPSC_MD). iPSC_Fb was established previously, and human primary bone marrow derived MSCs were reprogrammed in this study (FIG. S1).

[0060] These MSC derived iPSCs (iPSC_MD) were characterized with different specific markers, including stem cell surface markers SEEA4, TRA-1-60 and TRA-1-81, as well as transcriptional factors SOX2 and OCT4 (FIG. 1A and FIG. S2). After Sendai virus was excluded at passages 11 in vitro (FIG. S3), the iPSC_MD cells were injected into C.B-17/Icr-scid-bg (SCID Beige) mice, and formation of well-encapsulated cystic tumors that harbored differentiated elements of all three primary embryonic germ layers was observed (FIG. 1B). All three germ layers can be found within embryoid bodies formed by iPSC_MD cells with corresponding marker genes expressed (FIGS. 1C and 1D). It was also checked that iPSC_MD cells had normal karyotypes using chromosomal microarray (FIG. 1E). These data indicated therefore that iPSCs cell lines have been established derived from hMSCs. One representative iPSC_MD cell line C16 was used for the following experiments.

Example 2. Derivation of MSC-Like Cells from Human iPSCs and ESCs (iMSCs)

[0061] Induced pluripotent stem cells can be further induced into MSC-like cells by inhibiting of TGF-β signaling pathway combined with growth factors EGF and bFGF. A simplified protocol was developed herein and ESCs (H7) and iPSCs (iPSC_MD and iPSC_Fb) were induced to differentiate into MSC-like cells (iMSCs). ESCs and iPSCs derived iMSCs were characterized with specific surface markers by flow cytometry after the 22-day differentiate protocol and all three iMSCs presented correct MSC surface marker profile, which were CD44.sup.+, CD90.sup.+, CD73.sup.+ and CD105.sup.+ while CD11b.sup.−, CD19.sup.−, CD45−, CD14.sup.−, HLA-DR.sup.− (FIG. 2A). GelTrex coated wells were used for the differentiation procedure. iMSCs derived from iPSC_MD (iMSC_iPSC_MD) presented much better cell morphology and higher CD44 level. CD44, a cell surface glycoprotein involved in cell adhesion, migration and intercellular interactions, was significant higher expressed in iMSC_iPSC_MD. Therefore, it was further investigated the survival rate and efficiency on cell passages, by comparing seeding cells on the GelTrex coated or non-Geltrex coated wells. With higher CD44 expressed, iMSC_iPSC_MD had higher survival rate and efficiency during passage (FIG. 2C).

[0062] To further compare gene expression profiles of the ESCs, iPSCs, and iMSCs involved in this study, transcriptomes of these cell lines were analyzed using RNAseq. He and hierarchical gene clustering shown iMSC_iPSC_MD cells had more similar profile to the primary MSCs (FIG. 3). Furthermore, the differentiation genes of ESCs, iPSCs derived from MSCs and fibroblasts, and their differentiated iMSCs and the primary MSCs were compared (FIG. 3).

Example 3. iMSC Secreted Functional Factors as Similar as Primary MSC

[0063] It is aimed to investigate the immune modulation effect of iMSCs_iPSC_MD on cytokine release and expression profiles (FIG. 3). In order to compare the protein profiles of secreted and cell lysate between primary MSCs and iMSCs, cells from the same donor were used to reduce the noise background as well as unexpected variants due to individual differences. Forty different soluble human proteins levels were detected by using Human XL cytokine array. Results showed that secretion profiles were similar between the primary MSCs and iMSCs. They were summarized into 3 groups (FIG. 4A-F), which were increasing release or expression (>1.5 fold) or decreasing release or expression (<0.65 fold) or relatively similar on release or expression (0.65<x<1.5 fold).

[0064] For released cytokines, 12 kinds of cytokines were enhanced releasing by iMSCs while 11 kinds of cytokines were reduced releasing by iMSCs. For cytokines in cell lystates, 20 kinds of cytokines were higher expressed while 11 kinds of cytokines were lower expressed by iMSCs.

[0065] iMSC presented similar cytokine profiles as primary MSCs. These cytokines are mainly relate to adhesion and migration molecules (CD105, ICAM, MCP-1, SDF-1, etc.), growth factors (GM-CSF, FGF, PDGF-AA, BDNF and HGF, etc.), immune cytokines (IL-6, IL-8, GRO-a, RANTES and IL17A, etc.), angiogenesis cytokines (VEGF, angiopoietin-2, Angiogenin and Chitinase 3-like 1) and osteogenesis cytokines (Osteopontin), which has been reported to be secreted by bone marrow MSCs before. It was also observed that the secretion profile was not exactly the same as the cell lysates profile, which indicated the potential of MSCs/iMSCs functions would be various depending on the stimulation and microenvironments. Therefore, it is necessary to examine the bioactivity of iMSCs on their immune-modulation effects.

[0066] HGF mediates mesenchymal stem cell-induced recovery in many diseases, such as multiple sclerosis in EAE mice [11] and ischemia/reperfusion-induced acute lung injury in rats [40]. HGF and its primary receptor cMet are critical in MSC-stimulated recovery in EAE, neural cell development and remyelination [11]. Active MSC-CM contained HGF, and exogenously supplied HGF promoted recovery in EAE. Therefore, the HGF level was further detected in the iMSC-CM and primary MCS-CM by human ELISA kit. It is found that about 10 pg/ml HGF was contained in iMSC-CM or MSC-CM (every 0.2×10{circumflex over ( )}6 cells in 2 ml culture medium). Furthermore, it is aimed aimed to investigate the functional potential of these CMs in an available animal model, for example, EAE mouse model.

[0067] CD44 is a multifunctional cell surface molecule involved in cell adhesion, proliferation, differentiation, migration, angiogenesis, presentation of cytokines, chemokines and growth factors. It is also found that CD44 neutralizing antibodies (Hermes-1 or IM7) can impact on reduction of HGF by both iMSCs and MSCs after 18 hours treatments. However, this effect was mild when two different neutralizing antibodies used together. It was observed that the cells adherent ability was lost if the neutralizing process cost more than 48 hours (data not shown). Therefore, the CD44 is an essential factor for MSC survival and proteins secretion.

Example 4. Treatments with iMSCs and MSCs Conditional Medium can Improve Experimental Autoimmune Encephalomyelitis (EAE) Mice Recovery

[0068] In order to investigate the immune modulating effect of iMSC, the culture supernatant was collected from iMSC and concentrated by centrifugation with filter devices. It is found that both iMSC-CM and MSC-CM can improve EAE mice recovery (FIG. 5) within 30 days. However, the equal amount of concentrated basal MSCs medium (DMEM-LG+10% FBS) caused mice severely pathogenic symptoms and death in 24 hours (n=4), which had to be euthanized according to the animal ethics. Therefore, it was not shown in statistically analysis figures.

[0069] This issue was also considered for the conditioned medium collected from iMSCs and MSCs. Therefore, a simple component size examination was performed by using NanoSight NS300 (Malvern Panalytical, UK) (FIG. S4 and S5). Since DMEM-LG with 10% FBS was used for iMSC and MSC culturing, there was a large amount of component with the size between 50-100 nm. It is believed there were many from the FBS serum. But according to the EAE mouse experiments, after injection of concentrated and filtered DMEM-LG with 10% FBS medium, the mice were all dead. Thus, the exosome involved in the FBS was not useful for EAE treatment at least. For PBS injection group, the result of PBS solution had a peak with a larger size which might be the undissolved crystal or other disposes. For the comparison of iMSCs and MSCs, the size of CMs are mostly similar but with some minor difference. iMSCs-CM after 500 times dilution had 3 major peaks at 93 nm, 199 nm and 281 nm. While MSCs-CM after 100 times dilution had 3 major peaks at 103 nm. 226 nm and 299 nm. According to this data, it is believed that iMSC-CM had a higher enriched extracellular vehicles (EVs) compared to primary MSCs. Due to our experimental results, this CMs benefit for this EAE treatment but it also required further investigation and deeply understanding on how this EVs to work on immunomodulation and how to control the secretion profiles ex vivo.

Material and Methods

Reprogram Bone Marrow-Derived MSCs (BM-MSCs)

[0070] Human primary bone marrow derived MSCs were cultured in DMEM-LG medium with 10% FBS within 8 passages. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (A16517, Thermo Fisher Scientific, USA) was used and the reprogramming fibroblasts (Feeder-Free) protocol from the product's user guide was modified for BM-MSCs. Multiplicities of infection (MOI) used were KOS MOI=2.5-10, hc-Myc MOI=2.5-10, hK1f4 MOI=1.5-6. The best MOI was KOS MOI=8.75, hc-Myc MOI=8.75, hK1f4 MOI=4.5 (FIG. 1). For each sample, 1×10{circumflex over ( )}5 cells were seeded in a well of a 6-well plate and reprogrammed. The BM-MSCs are seeded in DMEM-LG+10% FBS medium on day −2 and transduced cells are plated on Geltrex (Thermo, USA) coated plates on day 7. Gradient medium change was required for early reporgramming process. The best condition was Gradient 2 method: On day 9 change: 70% of (DMEM-LG with 10% FBS)+30% of E8 medium; on day 10 change: 30% of (DMEM-LG with 10% FBS)+70% of E8 medium; and on day 11 change: 100% of E8 medium (shown Figures S6 and S7). Reprogramming process was repeated with cells from four health donors independently (MD1, MD2, MD3 and MD4).

[0071] Comparison:

[0072] 1.75× virus concentration+Gradient 2 medium replacing: (>20 colonies)

[0073] Normal virus concentration+Gradient 2 medium replacing: (3 colonies)

[0074] Normal virus concentration Gradient 1 medium replacing: 1 colony

[0075] 1.75× virus concentration+Gradient 1 medium replacing: 2 colonies

Immunofluorescence Staining

[0076] For immunofluorescence staining, cells will be placed on the coverslip (12 mm for 24-well Deckglaser, 20643, Germany) coated with Geltrex (Thermo) cultured in 24-well and fixed with 4% PFA. Cells will be washed with rinse buffer (PBS with 0.2% BSA and 0.3% TritonX-100) for 2 min and incubated with blocking buffer (PBS with 5% BSA and 0.3% TritonX-100) for 2 h. After blocking, cells will be incubated with primary antibodies at 4° C. overnight. Mouse anti-human SSEA4 (MC-813-70, DSHB), mouse anti-human TRA-1-60 (41000, Invitrogen) and mouse anti-human TRA-1-81 (411100, Invitrogen); and rabbit anti-human Oct3/4 (A16555, Life Technologies) and rabbit anti-human SOX2 (481400, Invitrogen) were used. On the second day, the cells will be washed with rinse buffer for 3 times (15 min per time). Then incubate the secondary antibodies (Goat anti-mouse IgG Alexa 488, 1:800; Goat anti-rabbit IgG Alexa 647, 1:200; Thermo Fisher Scientific, US) for 2 h at room temperature. Afterwards, cells will also be incubated with DAPI (Cell Signaling Technology, US) for 5 min and rinsed for 3 times (5 min per time).

Teratoma Formation in SCID Mice

[0077] In order to evaluate and monitor the pluripotency of iPSCs, teratoma formation assay was applied on Prepared resuspending iPSC-MD cells in DPBS with 2× ROCK inhibitor Y-27632 (SCM075, Sigma-Aldrich) at a concentration of 1×10{circumflex over ( )}6 cells per 50 μL of DPBS. And added an equal volume of chilled and liquid Matrigel (354248, Corning) together. Then gently mixed by using 25-gauge sterilized syringe and kept on ice until subcutaneous injection. The final injection volume of each mouse should be approximately 100 μL.

Quantitative Real-Time PCR (qPCR) of EBs

[0078] The expressions of pro-inflammatory factors were detected including SOX1, OTX2, GBX2, PAX6, TBC GSC, MIXL, SOX17, FOXA2 and AFP in the EBs by using qPCR method. In brief, EBs at day 7 were collected from the induction according to E6 medium protocol (Thermo). Total RNA was extracted Trizol Reagents (Invitrogen, USA) following the manufacturer's instructions. RNA quantification and purity were analyzed with Nanodrop (Thermo). The complementary DNA were synthesized by reverse transcript using Reverse Aid First Strand cDNA synthesis kit (Thermo Fisher Scientific, Rockford, Ill., USA), Quantitative PCR was performed using the Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific) and detected by StepOnePlus Real Time PCR (Thermo). The relative quantification of gene expression was conducted by 2.sup.−ΔΔCt methods. Results were represented as relative fold changes normalized to an in-ternal control gene GAPDH.

Chromosomal Microarray (CMA) Analysis

[0079] Karyotyping analysis of iPSC_MD clone 19 and clone 16 were carried out by using chromosomal microarray (CMA) analysis as previous reported [41]. Genomic DNA (250 ng) which extracted from each sample was subjected to genome-wide copy number variation (CNV) and absence of heterozygosity (AOH) analyses using CytoScan 750k SNP array (Affymetrix, Thermo Fisher Scientific). DNA was subjected to a series of restriction enzyme digestion, ligation, amplification, fragmentation, and labelling before loading onto the array for hybridization at 56° C. in GeneChip Hybridization Oven 645 (Affymetrix) according to manufacturer's instruction. After 18 hours of hybridization, the array will be washed in GeneChip Fluidics Station 450 (Affymetrix) before being scanned by GeneChip Scanner 3000 7G (Affymetrix). Results were visualized using Chromosome Analysis Suite (ChAS) version 4.0 (Affymetrix) and independently examined by two trained clinical scientists.

Generation of iMSC from Human ESCs or iPSCs

[0080] For differentiation of human ESCs/iPSCs into iMSCs, 7×10{circumflex over ( )}4 cells were seeded on a well of 6-well plate coated with Geltrex (Thermo) containing DMEM/F12 (11320082, Thermo) with 10% FBS. Two methods were described as below and figures were shown in FIG. S6.

[0081] Method 1: (1) 20% knockout serum and 10 μM SB431542 (ab120163, Abcam) for 6 days. (2) Then replaced medium with DMEM/F12 with 10% knockout serum and 1 μM SB431542 for another 6 days. (3) Then cells were trypsinized for 5 minutes at 37° C. and 1×10{circumflex over ( )}6 cells were seeded on a Geltrex coated or non-coated tissue culture 10 cm dish containing 8 ml of DMEM/F12 with 10% knockout serum and 10 ng/ml bFGF (13256029, Thermo) and 10 ng/ml EGF (PHG0311, Thermo) for 10 days. (4) Then medium was changed into DMEM-LG with 10% FBS in the following days. Medium were all changed every two days.

[0082] Method 2: (1) 10% knockout serum and 10 μM SB431542 (ab120163, Abcam) for 6 days. (2) Then replaced medium with DMEM/F12 with 1% BSA and 1× Insulin-Transferrin-Selenium (ITS-G) solution with 1 μM SB431542 for another 6 days. (3) Then cells were trypsinized for 5 minutes at 37° C. and 1×10{circumflex over ( )}6 cells were seeded on a Geltrex coated or non-coated tissue culture 10 cm dish containing 8 ml of DMEM/F12 with 10% knockout serum and 10 ng/ml bFGF (13256029, Thermo) and 10 ng/ml EGF (PHG0311, Thermo) for 10 days. (4) Then medium was changed into DMEM-LG with 10% FBS in the following days. Medium were all changed every two days.

[0083] Method 1 and Method 2 were equally efficient, but the reagent cost in Method 2 was cheaper.

MSC Surface Marker Characterization by Flow Cytometry

[0084] Cells were harvested by trypsinization and washed with 2% FBS-PBS twice; 2×10{circumflex over ( )}5 cells were re-suspended in 100 μl 2% FBS-PBS and incubated with the conjugated antibody for 30 min at room temperature in the dark. Stained cells were then washed with 2% FBS-PBS twice and re-suspended in 350 μl PBS for flow cytometry analysis (LSRII, BD); 10,000 events were recorded for each sample and data were analyzed with Flowjo. Antibodies against the human antigens CD11b conjugated Pacific Blue, CD19 conjugated APC, CD45 conjugated PerCPCy5.5, CD44 conjugated FITC, CD14 conjugated Pacific Blue, CD34 conjugated FITC, CD90 conjugated PerCPCy5.5, CD73 conjugated APC, CD105 conjugated PE and HLA-DR conjugated APC (BD, USA).

Transcriptome Analysis

[0085] Total RNAs were extracted using Trizol reagent according to the manufacturer's instructions. Transcriptome analysis by RNA-seq was performed at Novogene (HK) Co., Ltd. Briefly, mRNAs were enriched using poly-T oligo-attached magnetic beads, fragmented, and reverse transcribed into cDNAs. Sequencing libraries were prepared with the cDNAs and 150 bp pair-end reads were generated using an Illumina platform. Clean reads were mapped to reference genome using Tophat v2.0.12 with mismatch=2. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. Hierarchical clustering was performed using the log10(FPKM+1) value. Differential expression analysis was performed with DEGSeq. Corrected P-value of 0.005 and log2(Fold change) of 1 were set as the threshold for significantly differential expression.

The Proteome Profile of iMSCs and Human Primary MSCs

[0086] Human XL Cytokin Array was used to detect relative expression levels of individual analytes (ARY022, R&D Systems). 2×10{circumflex over ( )}5 cells of iMSC-iPSC-MD1 and human primary MSCs MD1 were seeded on a well of 6-wells-plate in DMEM-LG supplemented with 2% FBS. Triplicate wells of each type of cells. For cell culture supernates, medium was collected after 48 hours and combined depending on cell types. For cell lysates, rinse cells with PBS and process the procedures following the manufacture protocol. For data analysis, the average signal (pixel density) of the pair of duplicate spots represented each analyte. Subtracted an averaged background signal from each spot. Used a negative control spots as a background value. Then compared corresponding signals on different arrays to determine the relative change in analyte levels between samples.

Active EAE Induction and iMSC/MSC Conditioned Medium Treatment

[0087] Female C57BL/6 N mice were immunized for active induction of EAE as our previous described [42]. Briefly, the mice were subcutaneously injected with 200 μg MOG.sub.33-55 in complete Freund's adjuvant (5 mg/ml, Sigma-Aldrich). Pertussis Toxin (200 ng, List Biological Laboratories) was injected intravenously twice on 0- and 2-days post-immunization (dpi.). The immunized mice were daily monitored with body weight measurement and clinical score evaluation. EAE symptoms were scored for clinical severity as follows: 0, no clinical signs; 0.5, partially limp tail; 1, paralyzed tail; 1.5, hindlimb paresis or loss in coordinated movement; 2, loss in coordinated movement and hindlimb paresis; 2.5, one hindlimb paralyzed; 3, both hindlimbs paralyzed; 4, hindlimbs paralyzed, weakness in forelimbs; 5, forelimbs paralyzed [43, 44].

[0088] Human primary MD1 and MD2 and iMSCs-iPSC-MD1 and iMSCs-iPSC-MD2 were all grown in DMEM-LG supplemented with 10% FBS for 10 days. The growth medium was collected and refreshed every two days. Combined growth medium of MD1 and MD2 while combined growth medium of and iMSCs-iPSC-MD1 and iMSCs-iPSC-MD2, which to ensure that the results were not donor specific. Blank conditioned medium was DMEM-LG with 10% FBS alone. All conditioned medium was concentrated 100-fold through 30K and 100K centrifugal filter devices (Amicon Ultra-15). 15 ml of either iMSC-CM or MSC-CM from three independent repeats were combined and concentrated with centrifugal filter devices with an Amicon Ultra-15 (30K and 100K). Previous report has proved that hepatocyte growth factor (HGF) mediates MSCs stimulated functional recovery in animal models of MS [11]. Therefore, HGF in each concentrated fraction of iMSC-CM and MSC-CM was detected and quantified before intravenous injections (Table 1). In particular, HGF-enriched iMSCs-CM and MSCs-CM (>100k) were prepared by using centrifugal filter devices (30K and 100K) and quantified by hHGF ELISA kit (n=4).

TABLE-US-00001 TABLE 1 The concentration of HGF/total protein in the concentrated medium >100K 30K < X < 100K <30K Total Total Total Protein HGF Protein HGF Protein HGF (ug/ml) (pg/ml) (ug/ml) (pg/ml) (ug/ml) (pg/ml) PBS 0.00 0.00 N.A. N.A. N.A. N.A. MSCs-CM 1985.36 236 840.11 1.27 560.93 0.23 iMSCs-CM 1471.43 729 1055.84 5.11 554.59 0.00 DMEM- 1875.11 0.00 N.A. N.A. N.A. N.A. LG + 10% FBS

[0089] Protein concentrations were estimated using the BCA Protein Assay Kit (23227, Thermo) and 3 mg/protein was used for in vivo treatments intravenously on day 18 at peak disease every other day for a total of three injections. Four groups of model mice had been included in, which were blank conditioned medium (n=3), PBS (n=5), iMSCs-CM (n=4) and MSCs-CM (n=4).

Histopathology

[0090] For H&E or Luxol fast blue (LFB) staining, mice (30 dpi) were perfused with PBS and then fixed with 4% paraformaldehyde (PFA). Isolated L4-L6 spinal cords were post fixed in 4% PFA overnight at 4° C., dehydrated in gradient ethanol, permeabilized with xylene, embedded into paraffin and cut into 5 μm sections. Slides were stained with H&E or LFB for assessment of inflammation and demyelination, respectively Inflammation and demyelination were scored as described previously [44]. Briefly, inflammation was scored as follows: 0, none; 1, a few inflammatory cells; 2, organization of perivascular infiltrates; and 3, increasing severity of perivascular cuffing with extension into the adjacent tissue; Demyelination was scored as follows: 0, none; 1, rare foci; 2, a few areas of demyelination; and 3, large (confluent) areas of demyelination.

Statistical Analysis

[0091] For comparison among multiple groups with one factor, statistical comparisons were made by one-way analysis of variance (ANOVA) following with multiple comparisons by using GraphPad Prism 6 (GraphPad software Inc, CA, USA). Each experiment was repeated for at least three times (n≥3). A P value of <0.05 was considered as statistically significant. Three different symbols were denoted as: *P<0.05, **P<0.01, and ***P<0.001. All values were expressed as mean±SEM.

[0092] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference herein in their entireties.

REFERENCE

[0093] 1. Hu, S., et al., Effects of cellular origin on differentiation of human induced pluripotent stem cell-derived endothelial cells. JCI Insight, 2016. 1(8). [0094] 2. Kyttala, A., et al., Genetic Variability Overrides the Impact of Parental Cell Type and Determines iPSC Differentiation Potential. Stem Cell Reports, 2016. 6(2): p. 200-12. [0095] 3. Sell, S., ed. Stem Cells Handbook. 2013, Springer Science & Buisiness Media. [0096] 4. Becker, A. J., C. E. Mc, and J. E. Till, Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature, 1963. 197: p. 452-4. [0097] 5. Friedenstein, A. J., R. K. Chailakhjan, and K. S. Lalykina, The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet, 1970. 3(4): p. 393-403. [0098] 6. Dominici, M., et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8(4): p. 315-7. [0099] 7. Deng, R., Law, A. H. Y., Shen, J., Chan, G. C., Mini Review: Application of Human Mesenchymal Stem Cells in Gene and Stem Cells Therapy Era. Current Stem Cell Reports, 2018. 4(4): p. 10. [0100] 8. Ankrum, J. A., J. F. Ong, and J. M. Karp, Mesenchymal stem cells: immune evasive, not immune privileged. Nat Biotechnol, 2014. 32(3): p. 252-60. [0101] 9. Kyurkchiev, D., et al., Secretion of immunoregulatory cytokines by mesenchymal stem cells. World J Stem Cells, 2014. 6(5): p. 552-70. [0102] 10. Galipeau, J. and L. Sensebe, Mesenchymal Stromal Cells: Clinical Challenges and Therapeutic Opportunities. Cell Stem Cell, 2018. 22(6): p. 824-833. [0103] 11. Bal, L., et al., Hepatocyte growth factor mediates mesenchymal stem cell-induced recovery in multiple sclerosis models. Nat Neurosci, 2012. 15(6): p. 862-70. [0104] 12. Kato, A., et al., Assignment of the human alpha 2-plasmin inhibitor gene (PLI) to chromosome region 18p11.1-q11.2 by in situ hybridization. Cytogenet Cell Genet, 1988. 47(4): p. 209-11. [0105] 13. Mihara, K., et al., Development and functional characterization of human bone marrow mesenchymal cells immortalized by enforced expression of telomerase. Br J Haematol, 2003. 120(5): p. 846-9. [0106] 14. Lian, Q., et al., Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation, 2010. 121(9): p. 1113-23. [0107] 15. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72. [0108] 16. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76. [0109] 17. Malik, N. and M. S. Rao, A review of the methods for human iPSC derivation. Methods Mol Biol, 2013. 997: p. 23-33. [0110] 18. Warren, L., et al., Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell, 2010. 7(5): p. 618-30. [0111] 19. Subramanyam, D., et al., Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat Biotechnol, 2011. 29(5): p. 443-8. [0112] 20. Anokye-Danso, F., et al., Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell, 2011. 8(4): p. 376-88. [0113] 21. Miyoshi, N., et al., Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell, 2011. 8(6): p. 633-8. [0114] 22. Kaji, K., et al., Virus free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 2009. 458(7239): p. 771-5. [0115] 23. Narsinh, K. H., et al., Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat Protoc, 2011. 6(1): p. 78-88. [0116] 24. Okita, K., et al., Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322(5903): p. 949-53. [0117] 25. Loh, Y. H., et al., Reprogramming of T cells from human peripheral blood. Cell Stem Cell, 2010. 7(1): p. 15-9. [0118] 26. Staerk, J., et al., Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell, 2010. 7(1): p. 20-4. [0119] 27. Wiley, L. A., et al., Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy. Curr Protoc Stem Cell Biol, 2017. 42: p. 4A 12 1-4A 12 14. [0120] 28. Megges, M., et al., Generation of an iPS cell line from bone marrow derived mesenchymal stromal cells from an elderly patient. Stem Cell Res, 2015. 15(3): p. 565-8. [0121] 29. Re, S., et al., Improved Generation of Induced Pluripotent Stem Cells From Hair Derived Keratinocytes—A Tool to Study Neurodevelopmental Disorders as ADHD. Front Cell Neurosci, 2018. 12: p. 321. [0122] 30. Boonkaew, B., et al., Establishment of an integration free induced pluripotent stem cell line (MUSIi005-A) from exfoliated renal epithelial cells. Stem Cell Res, 2018. 30: p. 34-37. [0123] 31. Zhang, S. Z., et al., Urine-derived induced pluripotent stem cells as a modeling tool for paroxysmal kinesigenic dyskinesia. Biol Open, 2015. 4(12): p. 1744-52. [0124] 32. Zhou, T., et al., Generation of human induced pluripotent stem cells from urine samples. Nat Protoc, 2012. 7(12): p. 2080-9. [0125] 33. Bilousova, G., et al., Osteoblasts derived from induced pluripotent stem cells form calcified structures in scaffolds both in vitro and in vivo. Stem Cells, 2011. 29(2): p. 206-16. [0126] 34. Villa-Diaz, L. G., et al., Derivation of mesenchymal stem cells from human induced pluripotent stem cells cultured on synthetic substrates. Stem Cells, 2012. 30(6): p. 1174-81. [0127] 35. Kagia, A., et al., Therapeutic Effects of Mesenchymal Stem Cells Derived From Bone Marrow, Umbilical Cord Blood, and Pluripotent Stem Cells in a Mouse Model of Chemically Induced Inflammatory Bowel Disease. Inflammation, 2019. 42(5): p. 1730-1740. [0128] 36. Khan, M. A., et al., iPSC-derived MSC therapy induces immune tolerance and supports long-term graft survival in mouse orthotopic tracheal transplants. Stem Cell Res Ther, 2019. 10(1): p. 290. [0129] 37. Yang, H., et al., Human induced pluripotent stem cell-derived mesenchymal stem cells promote healing via TNF-alpha-stimulated gene-6 in inflammatory bowel disease models. Cell Death Dis, 2019. 10(10): p. 718. [0130] 38. Cuascut, F. X. and G. J. Hutton, Stem Cell-Based Therapies for Multiple Sclerosis: Current Perspectives. Biomedicines, 2019. 7(2). [0131] 39. Guilak, F., et al., Designer Stem Cells: Genome Engineering and the Next Generation of Cell-Based Therapies. J Orthop Res, 2019. 37(6): p. 1287-1293. [0132] 40. Chen, S., et al., Hepatocyte growth factor-modified mesenchymal stem cells improve ischemia/reperfusion-induced acute lung injury in rats. Gene Ther, 2017. 24(1): p. 3-11. [0133] 41. D′Antonio, M., et al., High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells. Stem Cell Reports, 2017. 8(4): p. 1101-1111. [0134] 42. Li, W., et al., Radix Rehmanniae Extract Ameliorates Experimental Autoimmune Encephalomyelitis by Suppressing Macrophage-Derived Nitrative Damage. Front Physiol, 2018. 9: p. 864. [0135] 43. Stromnes, I. M. and J. M. Goverman, Active induction of experimental allergic encephalomyelitis. Nat Protoc, 2006. 1(4): p. 1810-9. [0136] 44. Wu, H., et al., Caveolin-1 Is Critical for Lymphocyte Trafficking into Central Nervous System during Experimental Autoimmune Encephalomyelitis. J Neurosci, 2016. 36(19): p. 5193-9. [0137] 45. Ksiazek, K., A comprehensive review on mesenchymal stem cell growth and senescence. Rejuvenation Res, 2009. 12(2): p. 105-16. [0138] 46. Trento, C. and F. Dazzi, Mesenchymal stem cells and innate tolerance: biology and clinical applications. Swiss Med Wkly, 2010. 140: p. w13121. [0139] 47. Ng, F., et al., PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood, 2008. 112(2): p. 295-307. [0140] 48. Sackstein, R., et al., Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat Med, 2008. 14(2): p. 181-7.