Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

Generation of MSC-like cells from human dermal fibroblasts using a chemical and growth factor cocktail

We used human primary dermal fibroblasts that have been passaged for less than 10 times in all our experiments. The schematic diagram illustrates the process (Fig. 1A). In the initial screen, we tested several different combinations with a panel of 30 candidate chemicals and growth factors selected from previous studies of cell conversion (Supplementary Table S1)¹⁷,¹⁸,¹⁹. SSEA-4 and PODXL were used as markers to isolate the potentially induced MSC-like cells with superior multipotency and expansion ability than the parental fibroblasts⁹,¹⁰,¹¹. A cocktail containing six chemical inhibitors and three growth factors (6C+3GF) that includes SP600125 (JNK inhibitor), SB202190 (p38 inhibitor), Go6983 (protein kinase C inhibitor), Y-27632 (ROCK inhibitor), PD0325901 (ERK1/2 inhibitor), CHIR99021 (GSK3β inhibitor), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) (6C+3GF) successfully generated SSEA-4^highPODXL^high MSC-like cells (Fig. 1B). An important characteristic of MSCs compared to fibroblasts is its high clonogenicity, defined by colony-forming unit-fibroblast, CFU-F²⁰,²¹. In a 96-well colony formation assay, CFU-F was measured by the negative linear relationship between the seeding density (cell number) and the incidence of no colony formation (% negative response)²⁰,²¹. The frequency of CFU-Fs for iMSCs was 1 per 7 cells, which was 16-fold higher than the fibroblasts (1 per 112 cells) (Fig. 1C), suggesting a much higher clonogenicity in iMSCs. The iMSCs and BMMSCs also exhibited much stronger clonogenicity than fibroblasts in the traditional colony-forming assay (Supplementary Figure S1).

iMSCs have similar molecular signatures to BMMSCs

We next investigated the molecular mechanism of the multipotency induced by our chemical cocktail. One of Yamanaka factors, the pluripotent transcription factor OCT4, has been indicated to contribute to the multipotency of MSCs²²,²³. OCT4 was up-regulated in iMSCs to a level similar to BMMSCs as compared to fibroblasts (Fig. 1D), suggesting that iMSCs may be induced through the action of OCT4.

To further compare the molecular signatures, microarray analysis was performed with three parental fibroblasts (a neonatal foreskin fibroblasts CRL2097 and two adult abdominal skin fibroblasts DF440547 and DF443480), three iMSCs from these fibroblasts, and BMMSCs. We also retrieved the expression information of one BMMSC from the publicly available Gene Expression Omnibus (GEO) database. Principal component analysis (PCA) of stemness genes revealed that iMSCs derived from dermal fibroblasts were similar to each other, while the parental fibroblasts and BMMSCs were quite different (Fig. 1E, Supplementary Table S2). Of note, these iMSCs were more similar to BMMSCs when compared to their parental fibroblasts (Fig. 1E). Gene Ontology (GO) analysis revealed that the genes up-regulated in iMSCs and BMMSCs were related to developmental process and cell differentiation, while the genes down-regulated were related to immune system process (Supplementary Figure S2). These data suggest that our chemical cocktail is able to convert fibroblasts to a multipotent cell state that is similar to BMMSCs at the molecular level.

The conversion of fibroblasts into SSEA-4^highPODXL^high cells is reproducible

The chemical cocktail (6C+3GF) consistently induced the conversion with an efficiency ranging from 26.4–64.0% in ten experiments (mean = 38%) (Supplementary Figure S3). These iMSCs could be maintained and expanded in regular culture medium in the absence of small molecules for at least 8 passages and still express SSEA-4 and PODXL (Supplementary Figure S4). Only the SSEA-4^highPODXL^high cells, but not SSEA-4^lowPODXL^low cells, were able to efficiently differentiate into osteoblasts and adipocytes (Supplementary Figure S5).

iMSCs express all traditional MSC markers

Next, we tested if these SSEA-4^highPODXL^high iMSCs express traditional MSC markers by flow cytometry. The marker expression profiles of iMSCs were nearly identical to BMMSCs (expressing CD90, CD44, CD73, and CD105 surface markers, but not CD11b, CD19, CD34, CD45, and HLA-DR) (Supplementary Figure S6), fulfilling the criteria of MSC markers defined by the ISCT⁵. In contrast to the homogenous marker expression in iMSCs, the expression of these markers was heterogeneous in different sources of fibroblasts. Compare to iMSCs and BMMSCs, foreskin fibroblasts (CRL2097) expressed low level of CD105 (Supplementary Figure S6A), while abdominal fibroblasts (DF443480) expressed low levels of CD90 and CD44 (Supplementary Figure S6B). Both of these two fibroblasts slightly expressed CD11, CD19, CD34, CD45 or HLA-DR (more profound in CRL2097), while these markers were negative in iMSCs/BMMSCs (Supplementary Figure S6). In summary, iMSCs have a similar surface marker profile to BMMSCs that is evidently different from the primary fibroblasts. The microarray (Fig. 1E) and surface marker data (Supplementary Figure S6) both suggest that our chemical cocktail can induce distinct fibroblasts to a more homogenized multipotent cell state.

iMSCs are multipotent cells similar to BMMSCs

To investigate if iMSCs are multipotent, we examined their ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Alkaline phosphatase (ALP) activity and Alizarin Red S staining (ARS) were examined to determine osteogenesis ability. ALP activity is required for bone formation in early osteogenesis; and ARS reveals the extent of calcium deposition, which is required for bone matrix formation in late osteogenesis. After osteogenic induction, iMSCs induced from fibroblasts CRL2097 exhibited ALP activity at day 10 and ARS at day 21 to an extent comparable to that of primary BMMSCs. In contrast, primary fibroblasts could not differentiate into osteoblasts (Fig. 2A,B and D). To determine adipogenesis ability, these iMSCs were cultured in adipogenic induction medium for 21 days, and the lipid drops were then stained with Oil Red O. BMMSCs and iMSCs, but not fibroblasts, efficiently formed lipid drops (Fig. 2C and D). Finally, for chondrogenesis, iMSCs and BMMSCs instead of fibroblasts efficiently differentiated into chondrocytes (Fig. 2E). The extent of chondrogenesis was judged by the presence of lacunae structures in cartilage revealed by hematoxylin-eosin staining and proteoglycans shown by Alcian blue staining (Fig. 2E). Thus, the chemical cocktail can induce fibroblasts CRL2097 to reprogram into functional multipotent iMSCs. We also examined iMSCs derived from dermal fibroblasts of two additional donors (DF440547 and DF443480) and again found that iMSCs exhibited the ability to further differentiate into osteoblasts, adipocytes, and chondrocytes to a degree comparable to BMMSCs (Supplementary Figure S7). Thus, our chemical cocktail can effectively convert fibroblasts into functional, multipotent MSC-like cells.

iMSCs, like BMMSCs, markedly decrease the fatality of endotoxin-induced acute lung injury in a mouse model and demonstrate anti-inflammation ability

Next, to examine if iMSCs can be as effective as BMMSCs in vivo, an acute lung injury (ALI) mouse model was performed (Fig. 3A). We found that intratracheal administration of iMSCs or BMMSCs 4 hours after lipopolysaccharide (LPS) treatment significantly repressed acute lung injury (Fig. 3B). Of note, in contrast to the 50% death observed in ALI mice treated with LPS and the solvent control, all mice injected with iMSCs or BMMSCs survived (Fig. 3C). These results were supported by histological evidence of acute lung injury and lung injury scores (Fig. 3B and D). In contrast, fibroblasts were not as effective as iMSCs and BMMSCs in the treatment of ALI (Fig. 3B–D). Thus, iMSCs, like BMMSCs, can inhibit LPS-mediated ALI in vivo. MSCs are known to possess immunomodulatory functions⁷,⁸,²⁴. To test whether iMSCs contribute to the repair via anti-inflammation, we examined the cytokine secretion levels. After 2 days of LPS treatment, the bronchoalveolar lavage fluid (BALF) was collected and multiplex cytokine assay was performed. The levels of pro-inflammatory cytokines IL-1β, TNF-α, and TNF-β were downregulated in iMSC- and BMMSC-treated mice to very similar levels (Fig. 4). Thus, iMSCs can treat the disease as effectively as BMMSCs possibly due to the similar anti-inflammation ability.

We also used GFP-labeled fibroblasts, iMSCs, and BMMSCs to track their in vivo localization at different time points and how they may contribute to the lung repair process. After the injection, we did detect GFP-labeled cells reside in the lung during the 4.5-hour-, 24-hour-, and 48-hour-period (Supplementary Figure S8A). We did not detect any GFP-labeled cells in the bone marrow by FACS (Supplementary Figure S8B), indicating that the lung-injected cells did not migrate to bone marrow within 48 hours. The GFP signal and the epithelial marker pan-cytokeratin (pan-CK) did not colocalize in the same cell (Supplementary Figure S8A), suggesting that fibroblasts/iMSCs/BMMSCs may not directly contribute to the alveolar tissues. Altogether, our results revealed the anti-sepsis effects of iMSCs and BMMSCs should be primarily based on their immunomodulatory functions.

Three to six chemicals are sufficient to generate iMSCs

Next, we examined whether all chemicals and growth factors (6C+3GF) are required for generating iMSCs. By adding one cytokine at a time, we found that all three growth factors, TGF-β, bFGF, and LIF, were dispensable for iMSC generation (Fig. 5A and Supplementary Figure S9), since six chemicals (6 C) were sufficient to generate iMSCs almost as efficiently as the chemical and growth factor cocktail (6C+3GF). The iMSCs derived from six chemicals (6 C) exhibited the ability to differentiate into osteoblasts (Fig. 5B) and adipocytes (Fig. 5C) as effectively as BMMSCs, suggesting that they are still multipotent. Finally, we tested if all six chemicals were required for the generation of iMSCs. Three chemicals, SP600125, SB202190, and Go6983, were sufficient to generate iMSCs, however at a much lower efficiency (Fig. 5A, condition 2). The removal of any one of these compounds from the six chemical cocktail (6C) reduced the efficiency of iMSC production (Fig. 5A).

Reagents

All culture media (unless otherwise specified) were purchased from Invitrogen (Carlsbad, CA, USA). All chemicals in the cocktails were purchased from LC Laboratories (Woburn, MA, USA), except Go6983, which was obtained from TOCRIS (Bristol, UK) (Supplementary Table S1). All recombinant proteins were purchased from Peprotech (Rocky Hill, NJ, USA) Supplementary Table S3). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

All experiments with primary human cells were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Human Subject Research Ethics, Academia Sinica (Taipei, Taiwan) (ID: AS-IRB03–105122). Human primary neonatal foreskin fibroblasts (CRL2097) started from passage 4 after isolation were purchased from the ATCC (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Media-high glucose (DMEM-HG) medium with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). Primary adult abdominal skin fibroblasts derived from two adult females (DF440547 and DF443480), who are 42 and 56 years old, respectively (LONZA, Basel, Switzerland). These cells were cultured in DMEM-HG with 10% FBS and started in culture from passage 1. Human primary bone marrow mesenchymal stem cells (BMMSCs) were purchased from LONZA and were cultured with Dulbecco’s Modified Eagle Media-low glucose (DMEM-LG) medium containing 10% FBS. All cells were cultured at 37 °C under 5% CO₂. All primary cells were cultured within 10 passages after isolation. The informed consent forms were obtained from all the subjects by ATCC and Lonza.

Generation of iMSCs

For iMSC generation, primary fibroblasts maintained for less than 10 passages were cultured in DMEM-HG for 2 days. The culture medium was then replaced with the chemical cocktail medium (6C+3GF) (Supplementary Table S3) or different combinations for 6 days. Then, cell sorting was performed. Cells were cultured at 37 °C under 5% CO₂. To obtain sufficient amounts of iMSCs for differentiation assay and mouse anti-sepsis assay, the cells were expanded to 6–8 passages.

Flow cytometry and cell sorting

For cell surface marker analysis, BMMSCs, iMSCs, and fibroblasts were incubated with FITC-conjugated anti-human SSEA-4 (clone MC-813-70; eBiosciences, San Diego, CA, USA) and PE-conjugated anti-human PODXL (clone B34D1.3; eBiosciences) antibodies. The cells were then analyzed by FACSCanto (Becton Dickinson, Franklin Lakes, NJ, USA). SSEA-4^high and PODXL^high cells were isolated using a cell sorter (FACS Aria^TM II, BD Biosciences). The data was further analyzed by FACSDiva software (BD Biosciences).

CFU-F negative linear relationship assay

In this assay, the decrease of seeding cell density increases the incidence of wells with no colonies. Sorted cells were seeded into 96-well plates containing 8 cell-dose groups: 0, 5, 10, 20, 40, 80, 160, 320 sorted cells per well. After incubation in optimal condition for 14 days, the wells with no colony were counted for each condition. The seeding cell number in which 37% of the wells are negative for colony formation is defined as the cell concentration of one CFU-F²⁰,²¹.

Microarray analysis

RNA was purified using an RNeasy kit (Qiagen, Hilden, Germany). Samples were prepared for array hybridization using the human Affymetrix 3’ IVT Express kit with GeneChip^® Human Genome U133A 2.0 chips following the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). The array results were analyzed using the Gene Spring GX 12.6 software (Agilent Technologies, Santa Clara, CA, USA). The microarray profiling of BMMSC_2 was described previously in the Pubmed GEO database (GSM1533333), and all other array data were uploaded to the Pubmed GEO database (GSE72693).

Osteogenic differentiation

Human primary BMMSCs and iMSCs were cultured in DMEM-LG medium with 10% FBS. Fibroblasts were cultured in DMEM-HG with 10% FBS. To induce differentiation, cells (1 × 10⁴ cells/cm²) were cultured with osteogenic induction medium (90% DMEM-HG, 10% FBS, 0.1 μM dexamethasone, 10 mM beta-glycerophosphate, and 0.05 mM L-ascorbic acid phosphate). The media were replaced twice per week during differentiation.

Alkaline phosphatase activity assay

After 10 days of osteogenic differentiation, cells were fixed with 4% PFA in PBS for 3 minutes. Alkaline phosphatase (ALP) staining was performed using alkaline phosphatase kits in accordance with the manufacturer’s instruction (Sigma-Aldrich). For the quantification of ALP activity, the cells were washed twice with PBS and incubated with the ALP substrate p-nitrophenyl phosphate (pNPP) at room temperature for 5–20 minutes. The absorbance at an optical density (O.D.) at 405 nm was measured.

Alizarin Red S staining

After 21 days of osteogenic differentiation, cells were fixed with ice-cold 70% ethanol at −20 °C for 1 hour and then washed with PBS. The cells were then stained with 40 mM Alizarin Red S (ARS) (pH 4.2) for 10 minutes and subsequently washed five times with ddH₂O before being air dried. For quantification, the cells were incubated with 1 mL of cetylpyridinium chloride buffer for 1 h to extract ARS, and the O.D. at 550 nm was then recorded.

Adipogenic differentiation

For adipogenic induction, cells were cultured in adipogenic induction medium (Biological Industries, Kibbutz Beit-Haemek, Israel), which was replaced twice per week during the 21-day differentiation period.

Oil Red O staining

Cells were fixed with 4% PFA for 1 hour, washed with 60% isopropanol, and then air dried. The lipid vesicles were stained with Oil Red O staining medium (30 ml of 0.5% oil red solution in 2-propanol diluted with 20 ml of water) for 10 minutes and then washed with distilled water. For quantification, Oil Red O was extracted with isopropanol, and the absorbance at 530 nm was measured.

Chondrogenic differentiation

For chondrogenic differentiation, BMMSCs, iMSCs, and fibroblasts (2.5 × 10⁵ cells) in separate 15-mL tubes were centrifuged at 500 g for 10 minutes. The pelleted cells were then incubated with chondrogenic induction medium (Biological Industries). The cells formed a spherical aggregate after overnight incubation. The cells were continuously induced for 21 days, and paraffin sections were taken to analyze the samples. After deparaffinization, the slides were stained with Hematoxylin-eosin or an Alcian blue solution.

Alcian blue staining

The pelleted cells were embedded in paraffin. After sectioning, slides were deparaffinized with xylene and hydrated with distilled water. After incubation of slides in 3% acetic acid for 3 minutes, the slides were stained with a 1% Alcian blue solution (in 3% acetic acid, pH 2.5) for 30–45 minutes. The slides were then washed with water for 2 minutes, dehydrated with xylene, and mounted with mounting solution (Thermo Fisher Scientific, Waltham, MA, USA).

Endotoxin-induced acute lung injury in mice

All animal experimental procedures and methods were performed in accordance with the relevant guidelines and regulations. All animal protocols were approved by Academia Sinica Institutional Animal Care and Utilization Committee (Protocol ID: 15–07–840). BALB/c female mice (6–8 weeks, National Laboratory Animal Center, Taipei) were first anesthetized with Tiletamine/Zolazepam (25 mg/kg) and xylazine (10 mg/kg) via an intraperitoneal route. Acute lung injury (ALI) was then induced by the intratracheal (i.t.) instillation of 40 mg/kg lipopolysaccharides (LPS) purified from E. coli O55:B5 (Sigma-Aldrich) or 100 μL PBS. At four hours after LPS treatment, mice were anesthetized again and then randomly divided into four groups: (1) PBS, (2) human fibroblasts (10⁶ cells in 100 μL PBS), (3) iMSCs (10⁶ cells in 100 μL PBS), and (4) human BMMSCs (10⁶ cells in 100 μL PBS). The survival of mice was followed for 48 h. The survival rate of each group was observed every 6 hours. For the histology analysis and lung injury analysis, the samples were collected before or at 48 hours post injection. The lung sections were fixed and stained. The whole lung area was scanned by Aperio ScanScope XT machine (Leica, Wetzlar, Germany). Then, Aperio ImageScope program (Leica) was used to analyze the total lung section under 4× zoom by adding 100 μm × 100 μm grid. Images of each lung section were further quantified by counting lung section area and inflammatory area. The injury area, which has alveolar and interstitial inflammation (immune cell number >5 in a 100 μm × 100 μm grid), was counted. The ratio of injury area in lung section is calculated as (injury area)/(lung section area). Injury score was determined by the following criteria: 0, no injury; 1, 25% injury; 2, 50% injury; 3, 75% injury; 4, 100% injury.

Multiplex bead array assay

To quantify the pro-inflammatory cytokine secretion levels, the antibody-coated beads in a high sensitivity MILLIPLEX kit (Merck Millipore, Darmstadt, Germany) was used in accordance with the manufacturer’s protocol. To measure the amounts of selected cytokines (IL-1β, TNF-α, and TNF-β), BALF from LPS-induced acute lung injury mice injected with PBS, fibroblasts, iMSCs, and BMMSCs respectively were collected. Then different interleukins antibody-coated beads were incubated with 100 μg total BALF protein for 16 hours and further analyzed by Luminex 200 (Merck)⁵⁵.

Statistical analysis

All statistical data are presented as the mean ± standard deviation (S.D.) of at least three biological replicates. Statistically significant differences were assessed by Student’s unpaired two-tailed t-test, where p-value < 0.05 was considered a significant difference.