6d). Open in a separate window Fig. defect, contributed to the robust osteogenic capability of FReP cells in a challenging clinically relevant traumatic scenario were confirmed by histological and immunohistochemical staining. Taken together, we have provided an extended potency, safety, and molecular profile of FReP cell-based bone regeneration. Therefore, FReP cells present a high potential for cellular and gene therapy products for bone regeneration. [15]. Moreover, transplanting pre-osteogenic initiated FReP cells in the muscle pouch of severe combined immunodeficiency (SCID) mouse led to Adapalene bone generation without tumor formation [15], which suggested that FReP cells could be used as a novel osteoprogenitor for bone regeneration. In the current study, we further improved the FMOD reprogramming technology. In addition, to further assess the potential of FReP cells in bone regeneration, we profiled the gene expression of FReP cells during osteogenesis and evaluated the osteogenic efficacy of FReP cells in a clinically relevant critical-sized calvarial defect model. 2. Materials and Methods 2.1. FMOD production cDNA of human FMOD transcript (Genbank assessor number: “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_002023″,”term_id”:”1519246452″,”term_text”:”NM_002023″NM_002023) was subcloned into a commercially available vector pSecTag2A (Life Technology, Grand Island, NY) with C-terminal His-tag and transfected into CHO-K1 cells (ATCC, Manassas, VA) [16]. After establishing a stable expression clone, the FMOD was produced and purified by a contract research organization GenScript (Piscataway, NJ). Briefly, Adapalene stable human recombinant FMOD-expressing CHO-K1 cell line was cultured in 1L serum-free Freestyle CHO Expression Medium (Invitrogen) at 37C, 5% CO2 in an Erlenmeryer flask. Cell culture supernatant was harvested on day 10 for purification with HiTrap? IMAC HP, 1-mL column (GE Healthcare, Uppsala, Sweden). The fractions from a 100 mM imidazole elution were collected and dialyzed against 20 mM phosphate-buffered saline (PBS), pH 7.4. After that, the sample with low conductivity was loaded onto HiTrap?Q HP 1-mL column (GE Healthcare) for further purification. The purified protein was then evaluated by SDS-PAGE and Western blot (Supplementary Fig. 1). FMOD purified under non-reducing conditions was dialyzed again and sterilized for cell reprogramming. 2.2. Cell Culture Human newborn foreskin BJ-fibroblasts (ATCC) were cultured in a 4:1 mixture of Dulbeccos Modified Eagles Medium (containing 4 mM L-glutamine, 1.0 g/L glucose and 1.5 g/L sodium bicarbonate; Life Technology) and Medium 199 (Life Technology), supplemented with 10% fetal bovine serum (FBS; Life Technology) and 1% penicillin/streptomycin (P/S; Life Technology). BJ-fibroblast-derived iPSCs (BJ-iPSCs) obtained by conventional retrovirus-mediated method [17] were maintained on Matrigel? hESC-qualified Matrix (BD Biosciences, San Jose, CA) pre-coated plates with mTESR?1 medium (STEMCELL Technologies, Vancouver, Canada). 2.3. FMOD reprogramming 4 105 cells/well BJ-fibroblasts were seeded in 6-well culture plates overnight to confluence before exposure to 0.4 mg/ml recombinant human FMOD in DMEM medium supplemented with 1% PS for reprogramming under a serum-free condition. Fresh medium was changed daily [15]. After 21-day continual FMOD reprogramming, FReP cells were harvested with ReLeSR? (an enzyme-free hESC and hiPSC selection and passaging reagent [18, 19]; STEMCELL Technologies), and cultured on Matrigel? hESC-qualified Matrix coated-plates with mTESR?1 medium [20]. 2.4. Embryoid body (EB) formation and characterization FReP cells harvested by ReLeSR? reagent were seeded on AggreWell? 800 Plates with AggreWell? medium (STEMCELL Technologies) for EB formation following the manufacturers instruction. After 3 days, EBs were harvested and cryostat sectioned at 5 m for immunological staining. 2.5. In vitro differentiation towards endoderm derivatives FReP cells harvested by ReLeSR? reagent were cultivated in RPMI 1640 medium (Life Technology) supplied with 2% FBS, 2 mM L-glutamine, 1% P/S, and 100 ng/ml recombinant activin A (R&D systems, Minneapolis, MN) for 4 days, and then cultured without activin A for an additional 8 days [15]. 2.6. In vitro osteogenic differentiation For osteogenesis, FReP cells and their parental BJ-fibroblasts were transferred to AF solution (Life Technology) pre-coated plates and cultured in osteogenic medium Adapalene [-Modified Eagles Medium Adapalene (Life Technology) supplied with 10% FBS, 50 g/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO), 10 mM -glycerophosphate (Sigma-Aldrich), 10?8 M dexamethasone (Sigma-Aldrich)and 1% P/S] for 4 weeks[15]. 2.7. Animal model All animal surgeries were performed under institutional approved protocols provided by Chancellors Animal Research Committee at UCLA (protocol number: 2008C084). 3 days prior Wisp1 to implantation, 5 105 tested cells were seeded on poly(DL-lactic-induction [15]. The detailed procedure of.