FIG. 4(A-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).

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 lysates, 20 kinds of cytokines were higher expressed while 11 kinds of cytokines were lower expressed by iMSCs.

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.

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.

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

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.

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) (FIGS. 9 and 10). 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)

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, hKlf4 MOI=1.5-6. The best MOI was KOS MOI=8.75, hc-Myc MOI=8.75, hKlf4 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 reprogramming 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 FIGS. 11 and 12). Reprogramming process was repeated with cells from four health donors independently (MD1, MD2, MD3 and MD4).

Comparison:

- 1.75× virus concentration+Gradient 2 medium replacing: (>20 colonies)
- Normal virus concentration+Gradient 2 medium replacing: (3 colonies)
- Normal virus concentration Gradient 1 medium replacing: 1 colony
- 1.75× virus concentration+Gradient 1 medium replacing: 2 colonies
  
  Immunofluorescence Staining

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

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

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, IL, 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^−ΔΔCtmethods. Results were represented as relative fold changes normalized to an in-ternal control gene GAPDH.

Chromosomal Microarray (CMA) Analysis

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 750 k 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

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. 11.

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.

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.

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

MSC Surface Marker Characterization by Flow Cytometry

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

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 log 10(FPKM+1) value. Differential expression analysis was performed with DEGSeq. Corrected P-value of 0.005 and log 2(Fold change) of 1 were set as the threshold for significantly differential expression.

The Proteome Profile of iMSCs and Human Primary MSCs

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

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_33-55in 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].

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 (>100 k) were prepared by using centrifugal filter devices (30K and 100K) and quantified by hHGF ELISA kit (n=4).

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

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

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

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.

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.

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