Subject(s)
Antifungal Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Hand-Foot Syndrome/diagnosis , Tinea/diagnosis , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/surgery , Colonic Neoplasms/drug therapy , Female , Hand-Foot Syndrome/etiology , Humans , Middle Aged , Tinea/drug therapy , Uterine Neoplasms/drug therapy , Uterine Neoplasms/surgeryABSTRACT
The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.
Subject(s)
Adipocytes, Brown/metabolism , Adipocytes, White/metabolism , Cell Differentiation/genetics , Pluripotent Stem Cells/metabolism , Transgenes/genetics , 3T3 Cells , Adipocytes, Brown/cytology , Adipocytes, White/cytology , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/cytology , Adipose Tissue, White/metabolism , Animals , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cells, Cultured , Cluster Analysis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Profiling , HEK293 Cells , Humans , Immunohistochemistry , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , PPAR gamma/genetics , PPAR gamma/metabolism , Pluripotent Stem Cells/cytology , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism , Transplantation, HeterologousABSTRACT
Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.
