X Chromosome Inactivation and Differentiation Occur Readily in ES Cells Doubly-Deficient for MacroH2A1 and MacroH2A2

Macrohistones (mH2As) are unusual histone variants found exclusively in vertebrate chromatin. In mice, the H2afy gene encodes two splice variants, mH2A1.1 and mH2A1.2 and a second gene, H2afy2, encodes an additional mH2A2 protein. Both mH2A isoforms have been found enriched on the inactive X chromosome (Xi) in differentiated mammalian female cells, and are incorporated into the chromatin of developmentally-regulated genes. To investigate the functional significance of mH2A isoforms for X chromosome inactivation (XCI), we produced male and female embryonic stem cell (ESC) lines with stably-integrated shRNA constructs that simultaneously target both mH2A1 and mH2A2. Surprisingly, we find that female ESCs deficient for both mH2A1 and mH2A2 readily execute and maintain XCI upon differentiation. Furthermore, male and female mH2A-deficient ESCs proliferate normally under pluripotency culture conditions, and respond to several standard differentiation procedures efficiently. Our results show that XCI can readily proceed with substantially reduced total mH2A content

Functional implications of macrohistones in embryonic stem cells and differentiated progeny

Chromatin properties are increasingly gaining interest as important factors in the maintenance of the pluripotent nature of ESCs, as well as a determinant factor in lineage specification during differentiation. Histone variants, specialized isoforms of canonical core histones that confer distinct functional properties to the nucleosomes, are thought to have significant impact on these fundamental processes. Macrohistones (mH2As) belong to the family of closely related proteins that is comprised of three subtypes; mH2A1.1 and mH2A1.2 are splice variants of the gene H2afy, while mH2A2 is encoded by a separate gene (H2afy2), located on a different chromosome. Protein products of these two genes have been found enriched on the inactive X chromosome and other heterochromatic regions, findings implicating mH2As in gene repression. Microarray expression analysis in mH2A1.2-deficient ESCs demonstrated significant regulation for the subset of genes associated with early cell lineage decisions. Approximately 1/3 of the regulated genes were down-regulated, in contrast to the proposed repressive function for mH2As. Chromatin immunoprecipitation sequencing (ChIP-Seq) studies revealed the presence of mH2A1-nucleosomes in the chromatin of male ESCs on all autosomes, while the sex chromosomes were markedly devoid of mH2A1 signals across their entire lengths. RNAi-induced depletion of the mH2A-pool in male and female ESCs did not lead to defects in growth rates or developmental potential of these cells either in vitro or in vivo. No difference was observed for ESCs that retain mH2A1.1 in the differentiated state, compared to ESCs that are depleted for all mH2A isoforms. Imprinted expression of a selected subset of genes was unaffected in female knockdown ESC lines. Results the presented here imply an important function for mH2A1 in the control of developmentally-regulated genes in ESCs, but indicate the presence of multiple epigenetic mechanisms that the can compensate for the function of mH2As during in vitro development. ^

Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a “hemogenic endothelium” (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45−) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources

Cholesterol-secreting and statin-responsive hepatocytes from human ES and iPS cells to model hepatic involvement in cardiovascular health.

Hepatocytes play a central and crucial role in cholesterol and lipid homeostasis, and their proper function is of key importance for cardiovascular health. In particular, hepatocytes (especially periportal hepatocytes) endogenously synthesize large amounts of cholesterol and secrete it into circulating blood via apolipoprotein particles. Cholesterol-secreting hepatocytes are also the clinically-relevant cells targeted by statin treatment in vivo. The study of cholesterol homeostasis is largely restricted to the use of animal models and immortalized cell lines that do not recapitulate those key aspects of normal human hepatocyte function that result from genetic variation of individuals within a population. Hepatocyte-like cells (HLCs) derived from human embryonic and induced pluripotent stem cells can provide a cell culture model for the study of cholesterol homeostasis, dyslipidemias, the action of statins and other pharmaceuticals important for cardiovascular health. We have analyzed expression of core components for cholesterol homeostasis in untreated human iPS cells and in response to pravastatin. Here we show the production of differentiated cells resembling periportal hepatocytes from human pluripotent stem cells. These cells express a broad range of apolipoproteins required for secretion and elimination of serum cholesterol, actively secrete cholesterol into the medium, and respond functionally to statin treatment by reduced cholesterol secretion. Our research shows that HLCs derived from human pluripotent cells provide a robust cell culture system for the investigation of the hepatic contribution to human cholesterol homeostasis at both cellular and molecular levels. Importantly, it permits for the first time to also functionally assess the impact of genetic polymorphisms on cholesterol homeostasis. Finally, the system will also be useful for mechanistic studies of heritable dyslipidemias, drug discovery, and investigation of modes of action of cholesterol-modulatory drugs

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic Progenitor Function and Overcomes Early In Vivo Transplantation Failure

Summary: Human pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs. : Bhatia and colleagues reveal that human PSC-derived hematopoietic progenitor cells fail to survive even 24 hr following in vivo bone marrow transplantation, while these same progenitors survive and proliferate for weeks in vitro. They link these observations to deficiencies in CXCR4 signaling, which, when rectified, lead to enhanced progenitor function and survival in the bone marrow and a transcriptional shift toward somatic hematopoietic stem cells gene profiles. Keywords: human pluripotent, hematopoietic stem cells, progenitors, bone marrow, transplantation, chemokine receptor, cell signalin

Reprogramming of normal human dermal fibroblasts.

<p>(A) Expression of the pluripotency markers NANOG and TRA1-60 as detected by immunofluorescence. Expression of NANOG (red) and TRA1-60 (green) was analysed in human embryonic stem cells (WA09) and human induced pluripotent stem cells (WK1 and WK6). Also shown is hDF1, the primary dermal fibroblast line that yielded WK1 upon OCT4, SOX2, KLF4 and c-MYC- mediated iPS. (B) Quantitative analysis by flow cytometry of Tra1-60 and SSEA4 cell surface marker expression in WA09, WK1 and WK6 cells. Cell were grown under feeder free conditions and cell surface markers detected using fluorescein-conjugated anti-human Tra1-60 and phycoerytherin-conjugated anti-human SSEA4 as described in the materials and methods before sorting. For graphical display, cell numbers for each bin were normalized to the bin containing the highest number of cells and plotted as a function of their relative fluorescence intensity (RFU). Histograms show both the untreated control (dotted line) and the treated cells (dashed line). (C) Quantitative analysis by flow cytometry of Tra1-60 and SSEA4 cell surface marker co-expression in WA09, WK1 and WK6 cells. Relative fluorescence intensities for SSEA4 are displayed as a function of the relative fluorescence intensities for Tra1-60 in the all cells. Untreated cells, (black dots) are displayed for identification of “non-fluorescent” cells, treated cells (grey dots) were identified through exclusion of non-fluorescent cells through gating into “non-fluorescent”, Tra1-60+ cells, SSEA4+ cells and Tra1-60+/SSEA4+ double positive cells. The numbers in each corner show the percentage of stained cells in the four gates. (D) Analysis of mRNA levels for the pluripotency markers OCT4 and REX1 in human pluripotent stem cells by qRT-PCR. No significant differences among the three pluripotent stem cell lines were observed. Error bars represent the standard error of the mean.</p

Induction of APO expression in HLCs derived from hESCs and hiPSCs.

<p>(A) Analysis of apolipoprotein A1, A2, A4, C3, E and LDLR mRNA expression by qRT-PCR (for cell-type nomenclature see Fig. 3A legend). Note that with the exception of APOB (LDL particles), APOC3 (VLDL particles) and APOE (all particles) all other apolipoproteins are part of HDL particles. Error bars represent the standard error of the mean. (B) Apolipoprotein expression by quantitative immunofluorescence. WK1_HLCs were labeled with anti-human ALB and either anti-human APOA1, APOA2, APOC3 or LDLR antibodies as described and analysed (see methods and materials). ALB expression was detected through a mouse anti-goat Alexa 594 conjugated secondary antibody (red) and the apolipoprotein expression was detected through a mouse anti-rabbit Alexa 488 conjugated secondary antibody. Insets depict representative high resolution images showing apolipoprotein (green) and albumin (red) expression in the top panels and DAPI (blue) in the bottom panels. Error bars represent the standard deviation.</p

Comparative analysis of gene expression levels in HepG2 cells, primary hepatocytes, liver and HLC derived from WK1, WK6 and H9 cells.

<p>Values for mRNA levels were expressed as fold β-actin mRNA amounts and then normalized to levels in primary hepatocytes.</p

Pluripotency analysis of human pluripotent cells by embryoid body and directed differentiation.

<p>(A) Spontaneous differentiation of human pluripotent stem cells through EB formation. Pluripotent stem cells (WA09, WK1 and WK6) were seeded onto ultra-low binding substrates (Becton Dickinson) and assayed for aggregation (see experimental procedures). Human iPS cells aggregated to EBs with a tempo and size comparable to EBs derived from WA09 hESCs. (B) Analysis by semi-quantitative RT-PCR of germ layer marker expression by EBs derived from WA09, WK1 and WK6 cells. EB populations were grown in triplicates and harvested after 21 days of aggregation for the preparation of single stranded cDNA from total RNA. Germ layer-specific markers were detected by PCR and subsequent agarose gel electrophoresis using primers for the neuroepithelial (ectoderm) markers NESTIN (NES) and NEUROPILIN 2 (NETO2), the endoderm markers SOX17 and α-FETOPROTEIN (AFP) and the mesoderm markers BRACHYURY (T), MATRILIN1 (MANTN1), KDR, and MYF5. GAPDH was used as a loading control. (C) Directed differentiation of human pluripotent cells into early endodermal, ectodermal and mesodermal cells as described (Materials and Methods). Expression of the early lineage markers OTX2 (ectoderm), BRACHYURY (T, mesoderm) and SOX17 (endoderm) was detected by immunofluorescence microscopy using fluorescein-conjugated secondary antibodies. Cell nuclei were detected using DAPI (blue).</p

Expression of pluripotency and definitive hepatocyte markers before and after iPS, and in HLCs derived from pluripotent cells.

<p>(A) Analysis of expression of the pluripotency markers OCT4 and REX1 by qRT-PCR in: undifferentiated hESCs (WA09_ESC) and stage 3B HLCs derived from WAO9 (WAO9_HLC); dermal fibroblast line hDF1, hiPSC line WK1 derived from hDF1 (WK1_iPS) and stage 3B hepatocyte-like cells derived from WK1 (WK1_HLC); and dermal fibroblast line hDF6, the hiPSC line WK6 derived from hDF1 (WK1_iPS) and stage 3B hepatocyte-like cells derived from WK6 (WK6_HLC). (B) Induction of expression of the hepatocyte markers ALB, AFP, and HNF4α by qRT-PCR before and after reprogramming, and after stage 3B differentiation (for cell-type nomenclature see Fig. 3A legend). (C) Cell counts for AFP and ALB expression in cytocentrifuged stage 3B cells. WK1_HLCs were labeled with anti-human ALB and anti-human AFP antibodies and quantified as described (see Experimental Procedures). The inset depicts a representative high magnification image showing AFP (green) and ALB (red) expression in the top panel and DAPI (blue) in the bottom panel. Error bars represent the standard error of the mean.</p