Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals. Undifferentiated aHDF- and PB-iPSCs from the same individuals (two Parkinson’s disease patients (PD #1 and PD #2) and one adult healthy donor (donor91))
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals. sibling aHDF-iPSC clones (201B6 and 201B7; derived from the same aHDFs) (1) undifferentiated state (n=4, biological replicate #1-#4) (2) CXCR4-positive cell populations sorted by flowcytometry after 7 days of endodermal differentiation (n=4, biological replicate #1-#4) (3) CXCR4-negative cell populations sorted by flowcytometry after 7 days of endodermal differentiation (n=4, biological replicate #1-#4)
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals.
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals.
Project description:Human induced pluripotent stem cells (hiPSCs) have the ability to differentiate into a variety of cells and to self-renew in vitro. Because of these two characteristics, hiPSCs have been expected to provide new applications for regenerative medicine/cell therapy. Although various in vitro differentiation protocols have been developed for the efficient derivation of specific cell types, hiPSC lines vary in their ability to differentiate into specific lineages. Therefore, surrogate markers that accurately predict the differentiation propensity of hiPSC lines could be helpful for the development and manufacture of hiPSC-derived cells for therapies and in vitro assays. Here, we tried to identify the marker genes that potentially predicts the differentiation propensity of hiPSCs into neural progenitor cells (NPCs). Using ten hiPSC lines, we searched for genes significantly correlated between expression levels in the undifferentiated state and neuronal differentiation efficiency using two differentiation induction methods, and selected genes that were commonly and predominantly correlated with neuronal differentiation. Among the genes correlated with NPC differentiation, we identified ROR2 as a novel predictive marker of NPC differentiation. ROR2 expression in hiPSCs correlates negatively with NPC differentiation capacity, and ROR2 knockdown enhances NPC differentiation. These findings suggest that ROR2 serves as a useful surrogate marker for selecting hiPSC lines appropriate for NPC differentiation.
Project description:We have generated human induced Pluripotent Stem cells (hiPSc) from two individuals with OPA1 haploinsufficiency, and one control donor, using Sendai virus-mediated delivery of reprogramming factors. hiPSc lines have been screened using SNP array to assess chromosomal stability (alongside the fibroblast lines from which they derived), and validation of the pluripotency of the hiPSc lines is provided by Pluritest assessment of transcriptome datasets, prior to differentiation to dopaminergic neuronal clutures and downstream functional assays. Mitochondrial fragmentation in iPSC-derived dopaminergic neurons with OPA1 haploinsufficiency underpins increased apoptosis and syndromic Parkinsonism.
Project description:The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell. (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EpiSCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR- 371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior.
Project description:Female human induced pluripotent stem cell (hiPSC) lines exhibit considerable variability in X-inactivation status. Some lines maintain one transcriptionally active X chromosome (Xa) and one inactive X (Xi) from donor cells. However, hiPSC lines that have two Xas are infrequently produced. We show here Xinactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated using the Kyoto method, which employs leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Lines derived on other feeders maintained an Xi. In addition, there appears to be a window in which SNL feeders promote Xi-reactivation. Upon differentiation, Xa/Xa hiPSCs silenced one X. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and inactivation. Gene expression patterns were compared between several human embryonic stem cell (hESC) and hiPSC lines. Gene expression ratios between genes on X and those on autosomes were calculated from each cell lines.
Project description:Female human induced pluripotent stem cell (hiPSC) lines exhibit considerable variability in X-inactivation status. Some lines maintain one transcriptionally active X chromosome (Xa) and one inactive X (Xi) from donor cells. However, hiPSC lines that have two Xas are infrequently produced. We show here Xinactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated using the Kyoto method, which employs leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Lines derived on other feeders maintained an Xi. In addition, there appears to be a window in which SNL feeders promote Xi-reactivation. Upon differentiation, Xa/Xa hiPSCs silenced one X. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and inactivation. Gene expression patterns were compared between several human embryonic stem cell (hESC) and hiPSC lines. Gene expression ratios between genes on X and those on autosomes were calculated from each cell lines.
Project description:The use of pluripotent stem cells in regenerative medicine and disease modeling is complicated by the variation in differentiation properties between lines. In this study, we characterized 13 human embryonic stem cell. (hESC) and 26 human induced pluripotent stem cell (hiPSC) lines to identify markers that predict neural differentiation behavior. At a general level, markers previously known to distinguish mouse ESCs from epiblast stem cells (EpiSCs) correlated with neural differentiation behavior. More specifically, quantitative analysis of miR-371-3 expression prospectively identified hESC and hiPSC lines with differential neurogenic differentiation propensity and in vivo dopamine neuron engraftment potential. Transient KLF4 transduction increased miR-371-3 expression and altered neurogenic behavior and pluripotency marker expression. Conversely, suppression of miR- 371-3 expression in KLF4-transduced cells rescued neural differentiation propensity. miR-371-3 expression level therefore appears to have both a predictive and a functional role in determining human pluripotent stem cell neurogenic differentiation behavior. [mRNA profiling (Illumina)]: Four human ESC lines (H9, I4, I6, HUES6) at undifferentiation stages were purified with stem cell surface marker SSEA4 and subjected to RNA extraction and hybridization on Illumina microarrays. Each sample has 3 biological repeats, one of which has two technical repeats. [miRNA profiling (Agilent)]: Four human ESC lines (H9, I4, I6, HUES6) at undifferentiation stages were purified with stem cell surface marker SSEA4 and subjected to RNA extraction and hybridization on Agilent microarrays. Each sample has 3 biological repeats, one of which has two technical repeats.