Association of the OPRM1 Variant rs1799971 (A118G) with Non-Specific Liability to Substance Dependence in a Collaborative de novo Meta-Analysis of European-Ancestry Cohorts

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Medial HOXA genes demarcate haematopoietic stem cell fate during human development.

Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34(+)CD38(-/lo)CD90(+)CD45(+)GPI-80(+) fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function

Expansion on Stromal Cells Preserves the Undifferentiated State of Human Hematopoietic Stem Cells Despite Compromised Reconstitution Ability

<div><p>Lack of HLA-matched hematopoietic stem cells (HSC) limits the number of patients with life-threatening blood disorders that can be treated by HSC transplantation. So far, insufficient understanding of the regulatory mechanisms governing human HSC has precluded the development of effective protocols for culturing HSC for therapeutic use and molecular studies. We defined a culture system using OP9M2 mesenchymal stem cell (MSC) stroma that protects human hematopoietic stem/progenitor cells (HSPC) from differentiation and apoptosis. In addition, it facilitates a dramatic expansion of multipotent progenitors that retain the immunophenotype (CD34+CD38−CD90+) characteristic of human HSPC and proliferative potential over several weeks in culture. In contrast, transplantable HSC could be maintained, but not significantly expanded, during 2-week culture. Temporal analysis of the transcriptome of the <em>ex vivo</em> expanded CD34+CD38−CD90+ cells documented remarkable stability of most transcriptional regulators known to govern the undifferentiated HSC state. Nevertheless, it revealed dynamic fluctuations in transcriptional programs that associate with HSC behavior and may compromise HSC function, such as dysregulation of <em>PBX1</em> regulated genetic networks. This culture system serves now as a platform for modeling human multilineage hematopoietic stem/progenitor cell hierarchy and studying the complex regulation of HSC identity and function required for successful <em>ex vivo</em> expansion of transplantable HSC.</p> </div

<i>Ex vivo</i> expanded CD34+CD38 −CD90+ cells demonstrate high preservation of the HSC transcriptional program.

<p>(A) Hierarchical clustering using spearman rank correlation and (B) principal component analysis comparing freshly isolated CD34+CD38−CD90+ cells (Day 0) to CD34+CD38+CD90− differentiated progenitors and to <i>ex-vivo</i> expanded CD34+CD38−CD90+ cells demonstrate high conservation of the HSC transcriptome during culture on OP9M2 stroma. (C) Number of genes with more than 2-fold change in gene expression compared to Day 0 CD34+CD38−CD90+ cells is shown. (D) Graph showing the relative expression of transcription factors known to regulate HSC development or maintenance. (E) Graph showing the relative expression of transcription factors known to initiate hematopoietic lineage commitment. Values represent a fold difference to freshly isolated CD34+CD38−CD90+ cells. Raw data is available for download from Gene Expression Omnibus (<a href="http://ncbi.nlm.nih.gov/geo" target="_blank">http://ncbi.nlm.nih.gov/geo</a>) (GSE34974).</p

<i>Ex vivo</i> expanded CD34+CD38 −CD90+ cells show temporal changes in transcriptional programs associated with HSPC function and cell adhesion.

<p>Fuzzy C cluster analysis identified two temporal gene expression patterns describing genes that were either (A) immediately downregulated upon culture or (B) downregulated progressively during culture. GO analysis of genes in (A) and (B) (shown below each cluster respectively) identified numerous transcription factors that were downregulated, either (C) immediately or (D) progressively during culture. Similarly, several cell adhesion molecules implicated in HSC-niche interactions were downregulated either (E) immediately or (F) progressively during the culture. Shown are fold difference in gene expression of CD34+CD38−CD90+ cells at each time point relative to Day 0 control CD34+CD38−CD90+ cells. CD34+CD38+CD90− indicates gene expression changes for differentiated progenitors relative to Day 0 control CD34+CD38−CD90+ cells.</p

Cultured human CD34+CD38 −CD90+ cells show shared gene expression changes with <i>Pbx1</i> deficient mouse HSC.

<p>Differential gene expression analysis was performed comparing HSC (LSKCD34-Flt3-) from control and <i>Pbx1</i> deficient mice (GEO series GSE9188). (A) Table showing the FDR-q values from the GSEA analysis on pairwise expression data comparing freshly isolated CD34+CD38−CD90+ human cells with each time point in culture to the differentially expressed genes from <i>Pbx1</i> deficient mouse LSKCD34-Flt3- cells. (B) Heat map showing 24 genes downregulated both in Pbx1 deficient LSKCD34-Flt3- cells and at least in one of the culture time points (right) (>1.5-fold with respect to freshly isolated CD34+CD38−CD90+ cells, FDR of 5%). (C) Heat map of 54 genes that were upregulated both in <i>Pbx1</i> deficient HSC and at least in one of the culture time points (left), (>1.5-fold with respect to freshly isolated CD34+CD38−CD90+ cells, FDR of 5%). (D) A summary figure illustrating the HSC properties that are preserved or lost in the presence or absence of supportive stromal cells.</p

The OP9M2 MSC stromal cell line supports long-term expansion of multipotent human CD34+CD38

<p>−<b>CD90+ cells.</b> Co-culture of human fetal liver CD34+ cells on OP9M2 stroma maintains cells with the characteristic CD34+CD38−CD90+ surface immunophenotype that HSC express. (A) Representative FACS plots of CD34+ fetal liver cells after co-culture either without stroma, on BFC012 stromal cells or on OP9M2 stromal cells are shown. (B) Representative FACS plots of fetal liver CD34+ cells co-cultured for 5 and 7 weeks on the OP9M2 stromal cell line are shown. (C) Upper chart shows total fold expansion of all hematopoietic cells from input CD34+ cells with or without OP9M2. Lower chart shows total fold expansion of CD34+CD38−CD90+ cells. Bold line represents 7 experiments (n = 7) while dashed lines represent 3 experiments (n = 3), as in some experiments HSPC cultured beyond 5 weeks had started to lose proliferative potential. (D) Fold expansion of CD34+CD38−CD90+ clonogenic progenitors (CFU-C, assessed in methylcellulose cultures) is shown. Error bars represent SEM (* p<0.05, n = 3). (E) Single cell assay documenting expansion of CD34+CD38−CD90+ cells that retain both myeloid and lymphoid differentiation potential is shown. 96-well plates were coated with OP9M2 stroma, and individual CD34+CD38−CD90+ fetal liver cells (freshly isolated or <i>ex vivo</i> expanded) were sorted directly into each well and cultured for 3 weeks to assess for recreating the CD34+CD38−CD90+ immature population and for myelo-lymphoid differentiation potential. (F) Total fold expansion of clonally multipotent progenitors in co-culture with OP9M2 is shown. Error bars represent SEM (* p<0.05, 2 weeks n = 3, and 5 weeks n = 1).</p

The OP9M2 stromal cell line supports maintenance of engraftable HSC.

<p>(A) Summary of total human reconstitution in the bone marrow of NSG mice 16 weeks post transplantation of all the progeny cells from 50,000 CD34+ input cells that were transplanted freshly (Day 0) or co-cultured for up to 7 weeks. Shown are all individual recipients and mean value. P value was calculated for each time-point in comparison to Day 0. (** p<0.01). (B) Representative FACS plot of bone marrow and spleen from NSG recipient mice showing long-term multilineage engraftment of 50,000 input CD34+ FL cells that were isolated freshly or co-cultured 2 weeks on OP9M2. (C) Comparison of engraftment capacity between purified CD34+CD38−CD90+, CD34+CD38−CD90-, CD34+CD38+CD90+ and CD34− cells 2 weeks after co-culture demonstrates that the engraftable HSC are maintained only in the CD34+CD38−CD90+ fraction. Each mouse was transplanted with the respective population derived from 50,000 input CD34+ human fetal liver cells in culture. Shown are all individual recipients and mean value from one experiment (** p<0.01, * p<0.05). (D) Limited dilution assays (n = 2) was performed to estimate NSG-RC frequency among fetal liver CD34+ cells that were transplanted freshly or after 2 week culture. Mice were transplanted with equal number of input CD34+ cells at different concentrations and analyzed 16 weeks post transplantation. No significant difference in NSG-RC number (Day 0∶1 in 2610, upper 4821, lower 1413 and 2 wk: 1 in 3003, upper 5953, lower 1515) (p = 0.77) was detected.</p

Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.

How can you combine professional-quality research with discovery-based undergraduate education? The UCLA Undergraduate Consortium for Functional Genomics provides the answe