Reprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells

Transcription

1 Stem Cell Reports, Volume 8 Supplemental Information Reprogramming of Dermal Fibroblasts into Osteo-Chondrogenic Cells with Elevated Osteogenic Potency by Defined Transcription Factors Yinxiang Wang, Ming-Hoi Wu, May Pui Lai Cheung, Mai Har Sham, Haruhiko Akiyama, Danny Chan, Kathryn S.E. Cheah, and Martin Cheung

2

3

4

5 Supplemental Figure Legends Figure S1. Screening for the optimal culture conditions and overexpression of KMS into mouse embryonic stem cells does not lead to the formation of SOX9-EGFP+/RUNX2+ nodules. Related to Figure 1. (A) Quantification of KMS-transformed nodules formed from d9 to 14 when cultured in conventional medium on SNL feeder. (B) Quantification of KMS-transformed nodules formed from d9 to 14 when cultured in hypoxic condition (2% O2). Data are expressed as means ± SD. 3 independent experiments are represented in A and B. (C) Retroviral transduction of KMS into mouse embryonic stem cells (ESCs) derived from Sox9-EGFP KI mice generated nodules expressing SOX9-EGFP from d14 to 16 compared to control without transduction in which none of the nodules expressed GFP and RUNX2 in all time points examined. Phase images show morphology of KMStransduced ESCs and control cells. Scale bar: 100µm. Figure S2. Generation of KMS-reprogrammed SOX9-EGFP+/RUNX2+ nodules by dox-inducible lentiviral system and screening for the minimal number of transcription factors for direct conversion of MDFs into SOX9-EGFP+/RUNX2+ nodules. Related to Figure 2. (A) MDFs transduced with dox-inducible lentiviral KMS vectors did not form nodules and/or express GFP and/or RUNX2 in all time points examined following 2 or 4 days of dox treatment whereas nodules began to form on day 10, expressed GFP and RUNX2 from d11 to 13 and maintained GFP but not RUNX2 expression on day 14 following 8 or 10 days of dox treatment. Insets in top right corner show phase images of transduced MDFs or nodules after dox treatment at the indicated time points. (B) Panels showing double immunofluorescence of anti-gfp and anti-runx2 on MDFs reprogrammed with KS, MS, KLF4, c-myc or SOX9 in mtesr medium from d10 to 14 and phase images of their corresponding transduced MDFs. (C) Micrographs of reprogrammed cells on d14. The scale bar represents 50 µm in A, 100 µm in B and C. Figure S3. Molecular characterization of KMS- and KM-reprogrammed SOX9-EGFP+/RUNX2+ nodules. Related to Figures 3 and 4. Real-time RT-PCR analysis of transcript levels for Sox9 (A), Runx2 (B), Col2a1 (C), Sox5 (D), Sox6 (E), CD9 (F), CD73 (G), Col10 (H), Osteopontin (I), Osteocalcin (J), Osterix (K), Cola1 (L), Ihh (M), Ppr (N), Gremlin 1 (O), Oct4 (P), Sox2 (Q), PPAry1 (R), Mmp3 (S) and Igf2 (T) in reprogrammed nodules with KMS or KM. mrna levels from each gene was normalized to Gapdh with fold change relative to MDFs. Data are expressed as means ± SEM. 3 independent experiments are represented in A-T. *p<0.05; **p< 0.01; ***p < 0.001

6 Table S1. List of primers used for real time RT-PCR amplification in this study. Related to Figures 3,4 and S3. Markers Forward primer 5-3 Reverse primer 5-3 Runx2 GGAGCTCGGCGGAGTAGTTC CTGTGGTTACCGTCATGGCC endo-sox9 AGCTCACCAGACCCTGAGAA TCCCAGCAATCGTTACCTTC ex-sox9 CTGGGAACAACCCGTCTACA CACCAGACCAACTGGTAATG Col2a1 TTCCACTTCAGCTATGGCGAT GACGTTAGCGGTGTTGGGAG Sox5 GACAGAAAGAGAATCCATTGGT TTCTTGATCAGCTCTTCCATCT Sox6 CTAAGAATGTCTTCCAAGCAAG AAGTAGTTTTTCCATGCAGGAG Ihh GGCTTCGACTGGGTGTATTA CGGTCCAGGAAAATAAGCAC Ppr ACAAAGGGTGGACGCCAGCA GCGGTCGCAGCGTCTGTAGG Col10a1 GCCAAGCAGTCATGCCTGAT GACACGGGCATACCTGTTACC Col1a1 GCAACAGTCGCTTCACCTACA CAATGTCCAAGGGAGCCACAT Osterix CGCTTTGTGCCTTTGAAAT CCGTCAACGACGTTATGC Osteocalacin CAGACACCATGAGGACCATC GGACTGAGGCTCTGTGAGGT Osteopontin CTTTCACTCCAATCGTCCCTA GCTCTCTTTGGAATGCTCAAGT Ppar-γ1 CCACCAACTTCGGAATCAGCT TTTGTGGATCCGGCAGTTAAGA Oct4 TCTTTCCACCAGGCCCCCGGCTC TGCGGGCGGACATGGGGAGATCC Sox2 TAGAGCTAGACTCCGGGCGATG TTGCCTTAAACAAGACCACGAAA A Mmp3 TGCTGTCTTTGAAGCATTTGGGT T GCACTTCCTTTCACAAAGACTCAG A Igf2 ACAACTTCGATTTGAACCACAT GAGAGCTCAAACCATGCAAACT TC Gapdh AGGTCGGTGTGAACGGATTTG TGTAGACCATGTAGTTGAGGTCA ex-klf4 CCCAGTGTGGTGGTACGGGAAAT GTCGTTGAACTCCTCGGTCT C ex-c-myc CCCAGTGTGGTGGTACGGGAAAT C GCTCGCTCTGCTGTTGCTGGTGATA G Gremlin1 AAGTGACAGAATGAATCGCACC GGACTGGGTCTGCTCAGAGT CD9 CTGGCATTGCAGTGCTTGCTA AACCCGAAGAACAATCCCAGC CD73 CAAATCCCACACAACCACTG TGCTCACTTGGTCACAGGAC endo: endogenous; ex: exogenous

7 Supplemental Experimental Procedures Immunofluorescence Transduced fibroblasts were cultured in 24-well plate and washed with PBS briefly before fixing in 4% paraformaldehyde (PFA) for 10 minutes. Cells were washed and permeabilized with PBS+0.1% Tween 20 (PBST) before blocking with 1% bovine serum albumin (BSA) in PBST for 30 mins at room temperature (R.T). Cells were then incubated with sheep anti-gfp (Ab-direct, ) and rabbit Anti-RUNX2 (Abcam, ab76956) diluted in blocking buffer at 4 C. After overnight incubation, cells were washed three times 20 mins each with PBST before incubating with anti-sheep-igg-fitc (Invitrogen, A11015) and anti-rabbit-igg-cy3 (Jackson Immuno Research Laboratories, Inc, ) for 2 hours at R.T. After washing three times in PBST, cells were mounted with DAPI (VECTASHIELD Hard SetTM) and photographed in an inverted fluorescence microscope (OLYMPUS BX51). Real Time RT-PCR RNA was extracted from reprogrammed nodules, MDFs, ESCs, P10 tibia growth plate, MSCs, osteoblasts, adipocytes and sorted SOX9-EGFP + cells using RNAspin Mini kit (GE Healthcare) and treated with DNase to remove any contaminating genomic DNA based on the manufacturer s protocol. 1µg of RNA was reversetranscribed with Superscript III Reverse Transcriptase (Invitrogen) and oligo(dt) 20 primer. For real-time PCR analysis, 2ul of cdna with 200nM primers was mixed with Power SYBR Green PCR Master Mix (Applied Biosystems) in a total reaction volume of 25µl. Real time PCR reaction was performed in a 96-well Optical Reaction Plate and run on the ABI StepOnePlus Real-Time PCR System with the following parameters: Holding stage: 95 C, 1 min. Cycling stage (40 cycles): 95 C, 15s; 60 C, 1 min. Melting curve stage: 95 C, 15s, 60 C, 1 min, 95 C, 15s. Each sample was run in triplicates and level of transcripts from each gene was normalized to endogenous Gapdh control. The primers used are listed in Table S1. In vitro differentiation For chondrogenic differentiation, reprogrammed SOX9-EGFP + /RUNX2 + nodules were manually picked and replated in chondrogenic differentiation medium consisting of high-glucose DMEM supplemented with 100 nm dexamethasone (Sigma), 50 µg/ml L-ascorbic acid 2-phosphate (Sigma), 40 µg/ml proline (Sigma), 100 µg/ml sodium pyuvate (Gibco), 1% penicillin/streptomycin and 50 mg/ml ITS-Premix (BD Biosciences) (Collaborative Biomedical; 6.25 ng/ml insulin, 6.25 mg transferrin, 6.25 ng/ml selenious acids, 1.25 mg/ml BSA) for 14 days before being processed for Alcian blue staining. For osteogenic differentiation, reprogrammed SOX9-EGFP + /RUNX2 + nodules were subjected to osteogenic differentiation medium containing high-glucose DMEM, 50 µg/ml L-ascorbic acid 2-phosphate, 10 mm β- glycerophosphate (Sigma), 10% FBS, and 1% penicillin/streptomycin for 14 days before being processed for Alizarin Red S staining. For adipogenic differentiation, reprogrammed SOX9-EGFP + /RUNX2 + nodules were incubated in DMEM with 10% FBS medium supplemented with 100 nm dexamethasone and 10 4 M L-ascorbic acid 2-phosphate for 14 days before being processed for Oil Red O staining. Alizarin Red S Staining Reprogrammed nodules were fixed in 10% formaldehyde in 1xPBS for 15 mins, stained with 40mM Alizarin Red S (Sigma) solution for 30 seconds to 5 minutes and washed 3 times with distilled water to remove excess staining solution. Alcian blue staining Reprogrammed nodules were fixed in 4% PFA for 10 mins at room temperature, washed with water twice followed by staining in the Alcian blue (Sigma) in 0.1N HCl solution for mins at room temperature. Stained cells were washed 3 times with water. Oil Red O staining Reprogrammed nodules were fixed in 4% PFA for 10 mins, washed with water twice followed by rinsing with 60% isopropanol before incubation with the freshly prepared Oil Red O working solution [0.3% Oil Red O (Sigma) in isopropanol] for 15 mins at room temperature. Stained cells were immediately rinsed with distilled water 4 times to remove excess Oil Red O solution.

8 Von Kossa staining Histological sections were deparaffinized, hydrated and incubated with 5% silver nitrate solution (Sigma) and placed under a 60-watt light bulb at a range of several inches for 30 mins. Sections were then washed with water 3 times, incubated with 5% sodium thiosulfate (BDH) for 2 mins to remove un-reacted silver and rinsed again in water. Nuclei were then lightly stained with Harris Haematoxylin for few seconds and excess stain was removed with water. Stained sections were dehydrated through graded ethanol, cleared in xylene (BDH) for 5 mins and mounted with Depex (BDH). Transplantation of KMS-derived Sox9-EGFP + /Runx2 + cells into the subcutaneous space of nude mice Reprogrammed Sox9-EGFP + /Runx2 + cells were suspended at cells/ml in mtesr and injected into the dorsal flanks of nude mice. After four weeks of injection, tissues were dissected, fixed in 4% PFA, dehydrated and embedded in paraffin wax. Serial histological sections were processed for immunofluorescence with SOX9 (Chemicon, AB5535), RUNX2 (Abcam, ab76956), OSTERIX (Abcam, ab94744), TYPE I COLLAGEN (Abcam, ab6308) and TYPE II COLLAGEN (Thermo, #MS-235-P0) antibodies, and hematoxylin-eosin or Von Kossa staining Transplantation of reprogrammed nodules into bone fracture Mice were given general anesthesia with 2% isoflurane inhalation and a lateral incision was made in the hind legs near the mid-length of the tibia bone. Under aseptic surgical conditions, animals received an open tibia fracture of the right hind limb by hand drill. 1 x 10 4 reprogrammed SOX9-EGFP + /RUNX2 + nodules were injected into the fracture site and the incision was closed with biological skin glue. Animals were allowed to recover spontaneously from anesthesia. Two doses of buprenorphine (0.1 mg/kg body weight) were used as analgesic, first during the procedure and then every 8-12 hours later. After surgery, mice were monitored daily by external examination and body weight was compared with control animals at the same age. After 6 days of transplantation, tibia bones were collected, fixed in 4% paraformaldehyde, decalcified with EDTA, processed and embedded in paraffin for immunofluorescence and hematoxylin-eosin staining.