Comparative analysis of human induced pluripotent stem cell‐derived mesenchymal stem cells and umbilical cord mesenchymal stem cells

Abstract Generation of induced pluripotent stem cells (iPSCs) and their differentiation into mesenchymal stem/stromal cells (iMSCs) have created exciting source of cells for autologous therapy. In this study, we have compared the therapeutic potential of iMSCs generated from urinary epithelial (UE) cells with the available umbilical cord MSCs (UC‐MSCs). For this, adult UE cells were treated with the mRNA of pluripotent genes (OCT4, NANOG, SOX2, KLF4, MYC and LIN28) and a cocktail of miRNAs under specific culture conditions for generating iPSCs. Our non‐viral and mRNA‐based treatment regimen demonstrated a high reprogramming efficiency to about 30% at passage 0. These UE‐iPSCs were successfully differentiated further into ectoderm, endoderm and mesoderm lineage of cells. Moreover, these UE‐iPSCs were subsequently differentiated into iMSCs and were compared with the UC‐MSCs. These iMSCs were capable of differentiating into osteocytes, chondrocytes and adipocytes. Our qRT‐PCR and Western blot data showed that the CD73, CD90 and CD105 gene transcripts and proteins were highly expressed in iMSCs and UC‐MSCs but not in other cells. The comparative qRT‐PCR data showed that the iMSCs maintained their MSC characteristics without any chromosomal abnormalities even at later passages (P15), during which the UC‐MSCs started losing their MSC characteristics. Importantly, the wound‐healing property demonstrated through migration assay was superior in iMSCs when compared to the UC‐MSCs. In this study, we have demonstrated an excellent non‐invasive and pain‐free method of obtaining iMSCs for regenerative therapy. These homogeneous autologous highly proliferative iMSCs may provide an alternative source of cells to UC‐MSCs for treating various diseases.

such as cord blood, bone marrow, adipose tissue or other connective tissues. 4 Currently, MSCs have been identified as a valuable cell source for therapy including the characteristics of immunomodulation, angiogenesis, anti-apoptosis, anti-fibrotic and chemo-attractive activities. 5,6 Moreover, the paracrine factors secreted from MSCs are facilitated to support the growth and differentiation of neighbouring cells to where it is transplanted. However, adult MSCs derived from majority of the sources have a limited proliferative capacity and with a heterogeneous cell population.
Among stem cells, MSCs are considered to have a wide range of therapeutical applications. MSCs have some unique biological abilities because of their immunomodulatory and regenerative therapeutic potentials. 7 MSCs also have the ability to modulate the humoral and cellular responses. 8 Furthermore, MSCs have the potential to secrete anti-inflammatory cytokines and chemokines which makes them suitable for treating autoimmune disorders. [9][10][11] Importantly, MSCs do not have class II antigen expression which is good for allogenic cell transplantation. 12 As interest grows, there are some critical issues with the use of MSCs for clinical treatment. First, MSCs were identified in bone marrow and later from several other sources. 4 Though adult MSCs can be obtained from various tissues, the number of cells available for therapy is still a major challenge, and the procedures for collecting the cells are highly invasive and painful.
In many countries, at during deliveries, the parents are advised  13 In this study, we have generated iPSC-derived MSCs from urinary epithelial cells (referred as iMSCs) which are isolated from human urine samples. Our novel non-invasive approach of generating iMSCs will be a good source of autologous cells for regenerative disease therapy.
With this simple promising non-invasive method, we have generated a high-quality, autologous iMSCs with a high replicative potential which are suitable for the regenerative therapy. Moreover, we compare the therapeutic efficiency of the generated human iMSCs with umbilical cord MSCs (referred as UC-MSCs).

| Institutional regulatory approval
This study protocol is approved by the UTHSC Institutional Review

| Urinary epithelial (UE) cell culture
We have collected urine sample after obtained written informed consent from a 55-year old male healthy volunteer. To isolate and culture the urinary epithelial (UE) cells from urine, we have employed a modified the protocol as described by others earlier. 14 Briefly, UE cells were isolated by centrifuging 150-200 ml of urine at 500 g for 10 min at room temperature. The pellet containing the UE cells was cultured in a 25-ml flask containing DMEM complete medium containing 15% FBS medium at 37℃ in a 5% CO 2 incubator with humidified air. The isolated UE cells were identified by the protein expression of CK19 and ZO1. When these cells become 80% confluent, they were sub-cultured and used for reprogramming experiments.
The human UC-MSCs were purchased from Sciencell Research Laboratories. The neonatal human foreskin fibroblasts (NUFF) from Stemgent, and human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC). These cells were cultured and maintained as per supplier's instruction.

| Non-viral reprogramming of human UE cellderived iPSCs
When the UE cells attained 80% confluent, they were sub-cultured into a 6-well plate coated with iMatrix (Reprocell USA Inc) with NS medium. Then, the UE cells were reprogrammed with the mRNA of OCT4, NANOG, SOX2, KLF4, MYC and LIN28 by using transfection agent, Lipofectamine along with a cocktail of microRNAs (Reprocell USA Inc) for 10 days as described in our earlier publications 15,16 From day 9, we have observed several iPSC-granulated colonies resembled human embryonic stem cell colonies. These iPSC colonies were identified by TRA1-60 live staining, and the positive colonies were manually picked and further cultured on Matrigel-coated plates in NS medium at 37℃ in a 5% CO 2 incubator with humidified air. These cells were used for further experiments.

| Alkaline phosphatase staining
The iPSCs were cultured in a 4-well dish for three days. The cells were washed twice with phosphate-buffered saline (PBS). Then, the cells were fixed with the fix solution for 2-5 min and again washed twice with PBS. Staining solution from the alkaline phosphatase kit (Stemgent, # 00-0055) was added to each well and incubated in the dark at room temperature for 5-15 min. The reaction was stopped by aspirating the solution and washing the wells twice with 2 mL of PBS. Stained colonies were observed under the microscope.

| Differentiation of iPSCs into endoderm cells
For the endoderm differentiation, UE-iPSCs were cultured in NS medium in a 30-mm culture dish. When the cells reached 70%-80% confluent, the NS medium was removed and replaced with the Stemdiff definitive endoderm medium (Stemcell Technologies) and cultured again for 14 days. We have observed that the culture displayed significant morphological changes including the cuboid cell shape of primary hepatocytes. These day 14 cells were used for qRT-PCR analysis for the mRNA expression of hepatocyte markers apolipoprotein A1 (APOA1) and α−fetoprotein (AFP) and immunofluorescence analysis for AFP protein expression.

| Differentiation of iPSCs into neuronal cells
For the differentiation of human UE-iPSCs into neuronal cells, the UE-iPSCs were cultured in NS medium in a 30-mm culture dish.
When the cells become 70%-80% confluent, the NS medium was removed and replaced with the neuronal induction medium and cultured again for 18 days (Stemcell Technologies). We have observed the changes in cell morphology and displayed the neuronal-like cells from day 14 onwards. The cells were collected on day 18 for the mRNA expression of neuronal-specific genes OLIG2 and MAP2 by qRT-PCR analysis and protein expression of Nestin by immunofluorescence analysis.

| Differentiation of iPSCs into mesoderm cells
For the differentiation of human iPSCs into mesoderm cells specifically endothelial cells (ECs), we used the protocol as described by us earlier. 15 Briefly, the iPSCs were plated in a 30-mm culture dish in NS medium. When the cells obtain 80% confluence, the cells were cultured in mesodermal medium (DMEM supplemented with 1X B27, 1X N2, 5 μM CHIR, 25 ng BMP4) for 3 days. Then, the cells were cultured in the StemPro34 medium for 4 days. The cells were allowed to grow in endothelial EGM2 medium until they became mature ECs. These ECs were further confirmed by the mRNA and protein analyses.

| Differentiation of human UE-iPSCs into iMSCs
For the differentiation of UE-iPSCs into iMSCs, we cultured and maintained UE-iPSCs in NS medium. When the cells reached 70%-80% confluency, the NS medium was removed and fresh mesenchymal induction medium (STEMdiff-ACF, Stem Cell Technologies) was added to the plates for 4 days followed by MesenCult ACF Plus medium for 21 days, during which the medium was changed once in every two days. These cells were further maintained and sub-cultured in MesenCult ACF plus medium in a 5% CO 2 incubator at 37℃. When the cells reached 80% confluent, they were subcultured using Typsin LE for further experiments.

| Differentiation of iMSCs into osteocytes
The iMSCs were seeded into a 30-mm culture dish at a density of 7.5 × 10 5 cells with the MesenCult ACF Plus medium. After 24 h, the MesenCult medium was removed, and 2 ml of osteocyte differentiation (OD) medium which contains alpha MEM medium supplemented with ascorbic acid (50 µg/ml), β-glycerophosphate (5 mM), 20% of FBS, 1% GlutaMAX and 1% of penicillin/streptomycin was added. After 7 days, the OD medium was replaced with osteocyte mineralization medium (OD medium with 10 nM dexamethasone) for another 14 days. The medium was changed once in every two days. After 21 days, the induced osteocytes (iOST) were collected and used for further mRNA and protein analyses.

| Differentiation of iMSCs into chondrocytes
The iMSCs were cultured in a 30-mm culture dish at a density of 7.5 × 10 5 cells in MesenCult ACF Plus medium. After 24 hours, the MesenCult medium was removed, and 2 ml of chondrocyte differentiation medium (Thermo Fisher) was added for 17 days. The medium was changed once in every two days. These induced chondrocytes (iCHON) were collected after day 17 and were characterized by mRNA and protein analyses.

| Differentiation of iMSCs into adipocytes
For the adipocyte differentiation, 7.5 × 10 5 iMSCs were seeded into a 30-mm culture dish with MesenCult ACF Plus medium. After 24 h, the MesenCult medium was removed, and 2 ml of adipocyte differentiation medium (Thermo Fisher) was added to the cells and was cultured for 11 days with the regular media change at every two days. These induced adipocytes (iADIPO) were collected after day 11 and were further characterized by mRNA and protein analyses.

| Quantitative RT-PCR analysis
To characterize the iPSCs and iMSCs, we have performed quantitative RT-PCR (qPCR) for studying the gene expression pattern as described in our earlier publications. 15 Briefly, the RNA was col-

| Western blot analysis
The Western blot analysis for the protein expression was performed in the UE-iPSCs and iMSCs as described in our earlier publications. 17,18 Briefly, the cells were collected and centrifuged for protein isolation. After centrifugation, 50 μl of lysis buffer was added to the cell pellet. Then, the samples were centrifuged at 12,000 g for 20 min at 4℃. The supernatant was carefully removed without disturbing the pellet. The isolated protein samples were quantified by Bradford's method using the AccurisTM instrument SmartReader 96-well microplate absorbance reader at 595 nm. Equal amount of proteins was calculated and loaded into a SDS-PAGE. After the electrophoresis is completed, the proteins from the gel were transferred into a PVDF membrane. Immunoblotting was performed using a specific primary and secondary antibody, followed by visualization of protein bands using LI-COR Phosphorimager (Odyssey) and analysed using the Image Studio Lite software.

| Flow cytometry analysis
Flow cytometry analysis was performed to characterize the UE-iPSCs and iMSCs phenotypes as described by us earlier. 19 Briefly, cells from a six-well plate were harvested and washed twice in

| Immunofluorescence staining
The immunofluorescence staining was performed to analyse the protein expression as described by us earlier. 15,18 Briefly, the cells reached 80% confluency, and they were washed with PBS and fixed using 4% paraformaldehyde for 3 min. The cells were rinsed with PBS for three times at one-minute internals of each wash.
Blocking was done using 5% donkey serum and then incubated with the primary antibodies for overnight at 4℃. On the following day, fluorescence-tagged secondary antibodies specific to the host and primary antibodies were added and incubated at 37℃ for 1 h.

Nuclear staining was done by using DAPI (Molecular Probes, Life
Technology), and the cells were covered by using a glass coverslip.
The stained slides were visualized using an inverted fluorescence microscope (OlympusIX71), and the images were captured using CellSens standard software.

| Scratch assay
First, the migration potentials of iMSCs and UC-MSCs were evaluated using scratch assay as described earlier. 20 Secondly, the migration capacity of condition medium from iMSCs and UC-MSCs was analysed by using human neonatal foreskin fibroblasts (NUFF) and human umbilical vein endothelial cells (HUVECs). These iMSCs, UC-MSCs, NUFF and HUVEC were cultured in their appropriate media.
Once the cells reached 70%−80% confluency, they were harvested, and a suspension of 20,000 cells was seeded on the culture inserts placed in 30-mm dishes. After 24 h, the inserts were removed, and this type of insert forms a homogeneous cell-free lane in the middle of the confluent monolayer of cells. Immediately after the removal of inserts, the resulting scratches were pictured under phase-contrast microscope and live imaging microscope (T0). After imaging, the cells were further cultured for another 24 h in the presence or absence of conditioned medium from UC-MSCs or iMSCs (T24). The cell migration between the scratch was video recorded using live imaging or imaged under phase-contrast microscope. The rate of cell migration was calculated using ImageJ software (NIH) by measuring the cell area covered in T0 and T24. The results were expressed in percentage of wound closure under specific conditioned medium.

| Transwell migration assay
Migration capacity of the UC-MSCs and iMSCs was evaluated using 24-well Transwell insert containing transparent polyester membrane having 8-micron pore size (Corning). A total of 75 × 10 3 cells per well were seeded in inserts of each Transwell plate in FBS-free media.
The wells hold the inserts, and 0.5 ml of DMEM supplemented with 10% FBS as attractant was added. The plates were incubated at 37℃ in a 5% CO 2 incubator for 48 h. The cells that migrated to the surface of the wells were stained with 0.5% crystal violet in 2% methanol for 20 min. The attached cells in the wells were washed in PBS for three times to remove excess dye. The number of stained cells was counted using an inverted phase-contrast microscope. The degree of migration was expressed as the number of migrated cells per 10× microscopic visual field (mvf).

| Karyotyping analysis
To determine the cell integrity and chromosomal abnormalities, G-banded karyotyping analysis was performed using 15 th passage iMSCs and UC-MSCs at the WiCell Inc, Madison, WI.

| The colony-forming unit (CFU) assay
The iMSCs and UC-MSCs were seeded into 30-mm culture dishes at a density of 6.25 ×

| Telomere length quantification
To analyse the absolute telomere length in different iMSCs, the cells were collected in different passages, and its DNA was isolated by DNeasy kit (Qiagen). The isolated DNA was analysed using Absolute Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories).

| Statistical analysis
All experiments were repeated at least three times. Results are presented as mean ± SD. Comparisons were performed by one-way ANOVA (GraphPad Prism), and probability values less than 0.05 were considered as statistically significant.

| Non-viral and safe method of reprogramming human UE cells into iPSCs
Human UE cells were isolated from the urine sample and characterized by the expression of proteins CK19 and ZO1 ( Figure S1A). These UE cells were cultured and maintained in a 25-ml cell culture flask containing complete DMEM. When these cells became 80% confluent, the cells were sub-cultured in a 6-well plate coated with iMatrix in NutriStem medium. At 70% cell confluency, the cells were transfected with pluripotent genes using a StemRNA Reprogramming kit for 10 days as described in our earlier publications. 15,16 The protocol we have used for reprogramming UE cells into iPSCs is represented in the schematic illustration ( Figure 1A). Phase-contrast microscopic images of sequential changes were observed on day 0, day 5, day 7 and day 9 during reprogramming of UE cells into iPSCs ( Figure S1B).
From day 9, we have observed that several iPSC-granulated colonies and cells containing large-size nuclei that were occupied the maximum area in the cytoplasm. The iPSC-positive colonies were identified by TRA1-60 live staining. The positive colonies were manually picked under the phase-contrast microscope and grown in new culture dishes ( Figure S1C). Furthermore, these iPSCs were confirmed by alkaline phosphatase staining ( Figure S1D). To quantify the reprogramming efficiency, we performed flow cytometry analysis and our data showed that 29.1% of cells were reprogrammed into iPSCs by expressing the important pluripotent proteins OCT4 and SOX2 ( Figure S1E) at passage 0 (P0). These UE-iPSCs that were above P7 were used for further experiments.

| Characterization of UE-iPSCs
Two colonies of UE-iPSCs were characterized; the qRT-PCR analysis data showed a significantly increased level of mRNA expression of pluripotent genes OCT4, NANOG and SOX2 when compared to nonreprogrammed UE control (UE-C) cells ( Figure 1B). The enhanced mRNA-specific pluripotent gene expressions were further corroborated by immunofluorescence staining of pluripotent proteins OCT4, SOX2 and SSEA4 ( Figure 1C). We also characterized the UE-iPSCs by Western blot analysis and confirmed that the reprogrammed UE-iPSCs were showing prominent protein bands for OCT4, SOX2 and NANOG ( Figure 1D). Our flow cytometric analyses further quantified that the UE-iPSCs showed more than 88% cells were expressing pluripotency proteins OCT4, NANOG and SOX2 ( Figure 1E).
The mRNA expression of telomerase reverse transcriptase (TERT), an enzyme which increases the length of telomeres, was found significantly increased in UE-iPSCs ( Figure 1F). This increased TERT expression demonstrated that the generated UE-iPSCs were having the potential of proliferation. Overall, our results strongly demonstrated that the iPSCs generated from the UE cells were truly proliferative human pluripotent stem cells. With this approach, we have generated six bona fide iPSC colonies and two iPSC colonies were used for further experiments.

| Confirmation of pluripotency by in vitro differentiation analysis
We have further studied the in vitro differentiation potentials of UE-iPSCs into mesoderm, endoderm and ectoderm lineage cells to prove the efficiency of their pluripotency. For the mesodermal lineage differentiation, we have cultured the UE-iPSCs in a mesoderm-specific culture medium for 21 days as described by us earlier. 15 Our qRT-PCR analyses demonstrated a significantly increased expression of endothelial cell (EC) genes CD31 and VE-cadherin in the mesodermal differentiated culture compared with the UE-iPSCs and UE-C cells (Figure 2A,B). Further, immunofluorescence analyses confirmed the expression of VE-cadherin protein in the EC differentiated culture ( Figure 2C). For the endodermal lineage differentiation, we have cultured the iPSCs in Stemdiff definitive endoderm-specific medium.
We have observed that the endoderm differentiation culture displayed significant morphological changes such as primary hepatocytes. The day 14 cells were collected, and qRT-PCR analysis was performed for the hepatocyte-specific markers apolipoprotein A1 (APOA1) and α-fetoprotein (AFP). Our qRT-PCR analysis demon-

| Generation and characterization of iMSCs derived from UE-iPSCs
We have examined the differentiation potential of UE-iPSCs into

| Trilineage differentiation potential of iMSCs
The trilineage differentiation potentials of iMSCs are the unique

| Comparison of iMSCs and UC-MSCs in relation to the expression of mRNA and proteins
In order to compare the MSC-specific gene and protein expression and CD45 ( Figure 5C,D). These results suggested that both cells are equally good in the expression of MSC-specific mRNAs and proteins.

| No significant difference in proliferation, growth and colony formation in iMSCs and UC-MSCs
Telomere length at the end of chromosomes ultimately defines the proliferative capacity of a cell. The status of telomeres is an important parameter for MSC quality, and the telomere lengths can be used in specific selection of the MSCs and can be used as a quality control measure to select the desired MSCs from a culture. 24 Hence, we have measured the distribution of telomere lengths in the iMSCs and UC-MSC cell population. To analyse the absolute telomere length in different MSCs, the iMSCs' early (P7) and late passages (P17) and UC-MSCs (P7) cells were collected and their DNAs were isolated. The isolated DNA was analysed using absolute human telomere length quantification qPCR assay as described in the Methods. Our results showed the telomere length was similar between iMSCs and UC-MSCs ( Figure 6A). Importantly, no significant difference in telomere length was observed between the early (P7) and late passages (P17) of iMSCs ( Figure 6A). The telomere length suggested that both MSCs have long telomere which can enhance their proliferative potential.
In order to analyse the chromosomal abnormalities, which may happen during the long culture of cells, we performed G-banded karyotyping analysis using iMSCs and UC-MSCs at their pas-  Figure 6D). The stained colonies were counted in at least 20 frames taken per dish at 4× magnification with EVOS microscope. We observed that there was no significant difference between iMSCs and UC-MSCs in the number of colonies formed ( Figure 6E).

| Anti-inflammatory properties of iMSCs and UC-MSCs
The anti-inflammatory cytokines are the immunoregulatory molecules that regulate the pro-inflammatory cytokines response. MSC that possess anti-inflammatory effects has been shown to have therapeutic advantages in preclinical studies. In order to study the anti-inflammatory potentials of MSCs, the constitutive expression of anti-and pro-inflammatory marker proteins was studied using iMSC and UC-MSC at the early (P7) and the late (P15) passages.
Gene expressions of major anti-inflammatory cytokines such as IL-  Figure 7F).

| iMSCs and its conditioned medium had a superior cell migration capacity than the UC-MSCs and its conditioned medium
Scratch assay is an in vitro method to assess the cell migration under specific culture condition. iMSC showed a significantly higher migratory capacity when compared to UC-MSC ( Figure 8A,B).
Similarly, the migratory effect of the conditioned medium from iMSCs showed significantly higher migration potential in both HUVECs ( Figure 8C,D) and NUFF cells ( Figure 8E,F) when compared to the conditioned medium from UC-MSCs. In addition, the Transwell migration assay showed that iMSC had significantly higher migratory capacity than UC-MSC ( Figure S4). These results clearly suggested that the iMSCs and its conditioned medium possessed a superior migration capacity in covering the scratch area as well as transmigrating ability through the porous membrane than the UC-MSCs and its conditioned medium.

| DISCUSS ION
MSCs are increasingly used for regenerative therapies under multiple disease conditions. Treatment with MSC is promising for various degenerative diseases mainly because of their multilineage   Mostly, UC-MSCs are used for allogenic therapy, and there are some possibilities for the development of an immune reaction. 38,39 In a randomized control study, systemic immune reactions such as increased plasma pentraxin-3, IL8 and TLR4 were observed in cerebral palsy patients treated with umbilical cord blood cells. 39  passage. Predominantly, IL-6 has been shown as a pro-inflammatory molecule, 46 and we have consistently observed that IL-6 mRNA expression is low in iMSC and UC-MSC. Currently, it is not clear why IL-1B expression is higher in iMSCs at the early passage (p7). However, a recent report indicated that priming of human MSCs with interleukin-1 induces them towards an anti-inflammatory and pro-trophic phenotype in vitro. 47 These results indicate that the generated iMSCs have enhanced anti-inflammatory, immunomodulatory and proliferative potentials even at the late passages. Furthermore, the migration potentials of the iMSC, UC-MSC and their conditioned medium were evaluated through scratch assay. The migration capacity of the iMSCs and its conditioned medium was significantly higher when compared to the UC-MSCs and its conditioned medium. These data in agreement with the previous findings with UC-MSCs 48 clearly suggested that the wound-healing property is better using iMSCs than UC-MSCs.
In this study, we have generated UE-derived autologous iMSCs, which are superior in maintaining the characteristics of MSC, at both the early and late passages, whereas the MSC characteris- In conclusion, we have developed non-invasive, safe, nonimmunogenic, autologous iMSCs, which are highly proliferating and maintaining its MSC characteristics without any chromosomal abnormalities even at the later passage. Our comparative study of iMSCs with UC-MSCs has shown that the iMSCs were equally good or even better than UC-MSCs for the same functions. Furthermore, iMSCs can provide an unlimited supply of cells which will be a suitable source of cells for developing clinical-grade autologous cells for therapy.

CO N FLI C T S O F I NTE R E S T
The authors declare no potential conflict of interest relevant to this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are available from the corresponding author upon reasonable request.