UM171 expands distinct types of myeloid and NK progenitors from human pluripotent stem cells.

Scaling up blood cell production from hPSCs is critical to advancing hPSC technologies for blood transfusion, immunotherapy, and transplantation. Here we explored the potential of the HSC agonist pyrimido-indole derivative UM171, to expand hematopoietic progenitors (HPs) derived from hPSCs in chemically defined conditions. We revealed that culture of hPSC-HPs in HSC expansion conditions (SFEM with added TPO, SCF, FLT3L, IL3 and IL6) in the presence of UM171 predominantly expanded HPs with a unique CD34+CD41aloCD45+ phenotype that were enriched in granulocytic progenitors (G-CFCs). In contrast, in lymphoid cultures on OP9-DLL4, in the presence of SCF, FLT3L, and IL7, UM171 selectively expanded CD34+CD45+CD7+ lymphoid progenitors with NK cell potential, and increased NK cell output up to 10-fold. These studies should improve our understanding of the effect of UM171 on de novo generated HPs, and facilitate development of protocols for robust granulocyte and lymphoid cell production from hPSCs, for adoptive immunotherapies.

GATA2 Is Dispensable for Specification of Hemogenic Endothelium but Promotes Endothelial-to-Hematopoietic Transition.

The transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the hemogenic endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2-/- hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin+CD43-CD73+ non-HE or VE-cadherin+CD43-CD73- HE generation and subsequent HE diversification into DLL4+ arterial and DLL4- non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages.

CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

Coordination of fibronectin adhesion with contraction and relaxation in microvascular smooth muscle.

AIMS: The regulation of vascular diameter by vasoconstrictors and vasodilators requires that vascular smooth muscle cells (VSMCs) be physically coupled to extracellular matrix (ECM) and neighbouring cells in order for a vessel to mechanically function and transfer force. The hypothesis was tested that integrin-mediated adhesion to the ECM is dynamically up-regulated in VSMCs during contractile activation in response to a vasoconstrictor and likewise down-regulated during relaxation in response to a vasodilator. METHODS AND RESULTS: VSMCs were isolated from the Sprague-Dawley rat cremaster muscles. Atomic force microscopy (AFM) with fibronectin (FN)-functionalized probes was employed to investigate the biomechanical responses and adhesion of VSMCs. Responses to angiotensin II (Ang II; 10(-6) M) and adenosine (Ado; 10(-4) M) were recorded by measurements of cell cortical elasticity and cell adhesion. The results showed that Ang II caused an immediate increase in adhesion (+27%) between the probe and cell. Cell stiffness increased (+70%) in parallel with the adhesion change. Ado decreased adhesion (-15%) to FN and reduced (-30%) stiffness. CONCLUSION: Changes in the receptor-mediated activation of the contractile apparatus cause parallel alterations in cell adhesion and cell cortical elasticity. These studies support the hypothesis that the regulation of cell adhesion is coordinated with contraction and demonstrate the dynamic nature of cell adhesion to the ECM. It is proposed that coordination of adhesion and VSMC contraction is an important mechanism that allows for an efficient transfer of force between the contractile apparatus of the cell and the extracellular environment.