Project description:It has been notoriously challenging for researchers in the fields of stem cell biology and regenerative medicine to robustly generate uniform populations of differentiated cells from human induced pluripotent stem cells (hiPSCs). In particular, the process of iPSC chondrogenesis has been difficult to reproduce among different laboratories. While several differentiation protocols are being developed, few, if any, yield homogenous hiPSC-derived chondrocytes without any off-target contaminating cells. To improve homogeneity of hiPSC chondrogenesis, we have developed the first application of single-cell RNA sequencing to systemically deconstruct each differentiation step and delineate molecular mechanisms underlying lineage commitment of hiPSCs toward chondrocytes. We identified that MITF as well as several specific canonical and non-canonical WNTs are hub genes of gene regulatory networks governing the off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. By inhibiting these off-target molecules, we significantly enhanced chondrogenic lineage commitment of hiPSCs without cell sorting. Using sequencing data of day 28 (pellet time point) as an example, the percentage of chondrocytes within the pellet was improved from 59.63% (standard protocol) to 87.92% (optimized protocol; note: the other 12.07% belong to mesenchymal cells). Importantly, none of the off-target neural cells and melanocytes were detected, suggesting 100% removal of off-target differentiation by the optimized method. Our optimized protocol was validated by 3 unique hiPSC lines and 3 unique hMSC lines, indicating the protocol can be broadly applied to multiple cell lines. Moreover, therapeutic efficacy of hiPSC-derived chondrocytes was further validated in vivo by subcutaneous implantation and osteochondral defect repair in immunodeficient mice. In addition to the 11 consecutive time points from mesodermal to chondrogenic differentiation stages that were sequenced by bulk RNA-seq, we also sequenced 14 chondrogenic time points at single cell resolution. We believe that our bioinformatic analyses including gene regulatory networks, multicellular signaling models, as well as the large amount of data reported here will provide invaluable resources to investigate cell fate decision during human cartilage development.

Project description:Cell-based models of many neurological and psychiatric diseases, established by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), have now been reported. While numerous reports have demonstrated that neuronal cells differentiated from hiPSCs are electrophysiologically active mature neurons, the âageâ of these cells relative to cells in the human brain remains unresolved. Comparisons of gene expression profiles of hiPSC-derived neural progenitor cells (NPCs) and neurons to the Allen BrainSpan Atlas indicate that hiPSC neural cells most resemble first trimester neural tissue. Consequently, we posit that hiPSC-derived neural cells may most accurately be used to model the early developmental defects that contribute to disease predisposition rather than the late features of the disease. Though the characteristic symptoms of schizophrenia SZ generally appear late in adolescence, it is now thought to be a neurodevelopmental condition, often predated by a prodromal period that can appear in early childhood. Postmortem studies of SZ brain tissue typically describe defects in mature neurons, such as reduced neuronal size and spine density in the prefrontal cortex and hippocampus, but abnormalities of neuronal organization, particularly in the cortex, have also been reported. We postulated that defects in cortical organization in SZ might result from abnormal migration of neural cells. To test this hypothesis, we directly reprogrammed fibroblasts from SZ patients into hiPSCs and subsequently differentiated these disorder-specific hiPSCs into NPCs. SZ hiPSC differentiated into forebrain NPCs have altered expression of a number of cellular adhesion genes and WNT signaling. Methods: We compared global transcription of forebrain NPCs from six control and four SZ patients by RNAseq. Results: Multi-dimensional scaling (MDS) resolved most SZ and control hiPSC NPC samples; 848 genes were significantly differentially expressed (FDR<0.01) Conclusions: The WNT signaling pathway was enriched 2-fold (fisher exact test p-value = 0.031). 1-2 independent differentiations (biological replicates) for each of four control and four schizophrenia patients were analyzed; samples were generated in parallel to neuron RNAseq data.

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MRNA from Ionizing radiation treatment of chondrocyte-like cells derived from human induced pluripotent stem cells: gene and microRNA expression profile and high-throughput screening of secreted cytokines, chemokines, and growth factors

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