Together, these findings demonstrate that by using a transcription factor-driven reprogramming approach we can efficiently generate osteo-chondrogenic cells that readily form bone in?vivo

Together, these findings demonstrate that by using a transcription factor-driven reprogramming approach we can efficiently generate osteo-chondrogenic cells that readily form bone in?vivo. Results Transient Formation of SOX9-EGFP/RUNX2-Expressing Cells during Chondrogenic Induction from Skin NF2 Fibroblasts by KLF4, c-MYC, and SOX9 To determine whether cells expressing SOX9 and RUNX2 (SOX9+RUNX2+), which marks osteo-chondroprogenitors, could be formed during the course of chondrogenic induction by KLF4, c-MYC, and SOX9 (hereafter abbreviated as?KMS), we performed retroviral-mediated expression of these factors in 2? 104 MDFs prepared from new given birth to knockin (KI) mice, which enabled us to monitor daily the expression of EGFP driven by over 14?days of culture Ticagrelor (AZD6140) in conventional medium (DMEM?+ 10% fetal bovine serum [FBS]), a time when cells acquire chondrogenic features based on previous studies (Physique?1A; Hiramatsu et?al., 2011). medium, mTeSR. KMS-reprogrammed cells possess gene expression profiles akin to those of native osteo-chondroprogenitors with elevated osteogenic properties and can differentiate into osteoblasts and chondrocytes in?vitro, but form bone tissue upon transplantation under the skin and in the fracture site of mouse tibia. Altogether, we provide a reprogramming Ticagrelor (AZD6140) strategy to enable efficient derivation of osteo-chondrogenic cells that may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. gene in limb bud osteo-chondrogenitors prior to the onset of chondrogenic? mesenchymal condensation resulted in a complete absence of cartilage and bone formation, whereas ablation of SOX9 function after mesenchymal condensation led to the impairment of chondrocyte proliferation and?differentiation, which was predominantly mediated by?the absence of and expression (Akiyama et?al., 2002, Smits et?al., 2001). In addition, the SOX trio Ticagrelor (AZD6140) regulates genes coding for the extracellular matrix components by binding to their enhancers (Bell et?al., 1997, Bridgewater et?al., 1998, Han and Lefebvre, 2008, Lefebvre et?al., 1998, Nagy et?al., 2011). Consistently, adenoviral-mediated expression of the SOX trio is sufficient to transform mouse dermal fibroblasts (MDFs) into chondrogenic cells expressing cartilage marker genes and secreting extracellular matrix. However, the induced cells still expressed fibroblast marker gene, gene expression by retroviral-mediated expression of two iPSC-reprogramming factors (c-MYC and KLF4) and a grasp regulator for chondrogenesis, SOX9 (Hiramatsu et?al., 2011). In?addition, this chondrogenic induction did not pass through a pluripotent state throughout the period of Ticagrelor (AZD6140) the reprogramming process (Outani et?al., 2011). Subsequently, the same research group generated induced chondrogenic (iChon) cells expressing type II but not type I COLLAGEN from human dermal fibroblasts with the same factors (Outani et?al., 2013). Both mouse and human iChon cells produced homogeneous cartilage-like tissues upon grafting in nude mice (Hiramatsu et?al., 2011, Outani et?al., 2013). However, it is not clear from these studies whether bipotential osteo-chondroprogenitors are generated during the direct conversion of fibroblasts into chondrocytes. Thus, it is conceivable that the ability to generate osteo-chondroprogenitors by?a reprogramming approach with defined factors may hold promise for cell replacement therapy not limited to cartilage but also for bone tissues. In the present study, we took advantage of this lineage-reprogramming approach to examine the possibility of osteo-chondroprogenitor formation using knockin (KI) reporter mice. We identified osteo-chondrogenic cells during the Ticagrelor (AZD6140) course of lineage reprogramming from skin fibroblasts to chondrocytes with gene expression profiles and in?vitro differentiation potency comparable with native osteo-chondroprogenitors in developing mouse limb bud. Transplantation of reprogrammed osteo-chondrogenic cells subcutaneously and into bone lesion site of immunodeficient recipients resulted in bone formation. Together, these findings demonstrate that by using a transcription factor-driven reprogramming approach we can efficiently generate osteo-chondrogenic cells that readily form bone in?vivo. Results Transient Formation of SOX9-EGFP/RUNX2-Expressing Cells during Chondrogenic Induction from Skin Fibroblasts by KLF4, c-MYC, and SOX9 To determine whether cells expressing SOX9 and RUNX2 (SOX9+RUNX2+), which marks osteo-chondroprogenitors, could be formed during the course of chondrogenic induction by KLF4, c-MYC, and SOX9 (hereafter abbreviated as?KMS), we performed retroviral-mediated expression of these factors in 2? 104 MDFs prepared from new given birth to knockin (KI) mice, which enabled us to monitor daily the expression of EGFP driven by over 14?days of culture in conventional medium (DMEM?+ 10% fetal bovine serum [FBS]), a time when cells acquire chondrogenic features based on previous studies (Physique?1A; Hiramatsu et?al., 2011). Thus, EGFP expression in transformed cells is considered to be an indicator of activation of transcription followed by RUNX2 immunofluorescence to determine co-expression of SOX9-EGFP and RUNX2. We found that transformed fibroblasts began to form aggregates and express EGFP as early as day 9 (d9) post transduction. By d10, initiation of RUNX2 expression was detectable in EGFP+ aggregates with distinct nodular appearance, became significant on d12 and d13, and diminished or were barely observed on d14, while EGFP was maintained from d9.