Variations of X Chromosome Inactivation Occur in Early Passages of Female Human Embryonic Stem Cells

X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs

Normal Human Pluripotent Stem Cell Lines Exhibit Pervasive Mosaic Aneuploidy

Human pluripotent stem cell (hPSC) lines have been considered to be homogeneously euploid. Here we report that normal hPSC – including induced pluripotent - lines are karyotypic mosaics of euploid cells intermixed with many cells showing non-clonal aneuploidies as identified by chromosome counting, spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH) of interphase/non-mitotic cells. This mosaic aneuploidy resembles that observed in progenitor cells of the developing brain and preimplantation embryos, suggesting that it is a normal, rather than pathological, feature of stem cell lines. The karyotypic heterogeneity generated by mosaic aneuploidy may contribute to the reported functional and phenotypic heterogeneity of hPSCs lines, as well as their therapeutic efficacy and safety following transplantation

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

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

GSK3 Deficiencies in Hematopoietic Stem Cells Initiate Pre-neoplastic State that Is Predictive of Clinical Outcomes of Human Acute Leukemia

Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3\u3b1 (GSK-3\u3b1) and GSK-3\u3b2 dependency leads to aggressive AML. Although GSK-3\u3b1 deletion alone has no effect, GSK-3\u3b2 deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3\u3b2 and GSK-3\u3b1 uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3\u3b2 provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3\u3b1- and GSK-3\u3b2-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution. Guezguez et al. show that progressive removal of glycogen synthase kinase-3 (GSK-3) signaling by Gsk3b allelic deletion results in an MDS state that, when combined with Gsk3a deletion, leads to AML. A molecular signature derived from Gsk3b-null cells has prognostic potential for MDS patients

Assessment of Climate Change Impact on Water Requirements of Orchards in Bosnia and Herzegovina

In agriculture, climate change will have the strongest impact on orcharding, due to the length of the growing season. High temperatures, droughts and more intense solar radiation could burn leaves and fruits, which would impair the quality and marketable yield. On the other hand, heavy rains, if they occur during the period of flowering and maturing, especially of cherries and berries (strawberries, blackberries, raspberries), would result in the spread of fungi, rotting of fruit and delayed picking. In this regard, the objective of the paper is to assess the extent to which climate change will affect the availability of water in traditional, rainfed orchards, as well as to promote adaptation initiatives. The CROPWAT 8.0 model was used to analyze water surplus and shortage. Input climate data pertained to a reference period and SRES climate scenarios A1B, A2 and RCP 8.5 applied to three characteristic climatic areas in Bosnia and Herzegovina. Analyses showed that water surplus would occur regularly in the colder part of the year, in each study area and by all scenarios. All study areas would also experience water shortages in summer, but with varying drought duration and severity. Adaptation measures are proposed for each study area and period of the year, such as the need for drainage in winter, irrigation in summer and application of new technologies for growing fruit trees