Sasidhar Vemula

Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony–forming cells

The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony–forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel–forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >108 ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.

ROCK1 functions as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability

Rho kinases belong to a family of serine/threonine kinases whose role in recruitment and migration of inflammatory cells is poorly understood. We show that deficiency of ROCK1 results in increased recruitment and migration of macrophages and neutrophils in vitro and in vivo. Enhanced migration resulting from ROCK1 deficiency is observed despite normal expression of ROCK2 and a significant reduction in overall ROCK activity. ROCK1 directly binds PTEN in response to receptor activation and is essential for PTEN phosphorylation and stability. In the absence of ROCK1, PTEN phosphorylation, stability, and its activity are significantly impaired. Consequently, increased activation of downstream targets of PTEN, including PIP3, AKT, GSK-3beta, and cyclin D1, is observed. Our results reveal ROCK1 as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation.

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1−/− and ROCK2−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.

Rho Kinase Regulates the Survival and Transformation of Cells Bearing Oncogenic Forms of KIT, FLT3, and BCR-ABL

We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3, and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene-bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient-derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth, and loss of actin polymerization in oncogene-bearing cells leading to significantly prolonged life span of leukemic mice. In summary we describe a pathway involving PI3K/Rho/ROCK/MLC that may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.

Genetic and cellular evidence of vascular inflammation in neurofibromin-deficient mice and humans.

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. NF1 patients display diverse clinical manifestations, including vascular disease, which results from neointima formation and vessel occlusion. However, the pathogenesis of NF1 vascular disease remains unclear. Vessel wall homeostasis is maintained by complex interactions between vascular and bone marrow-derived cells (BMDCs), and neurofibromin regulates the function of each cell type. Therefore, utilizing cre/lox techniques and hematopoietic stem cell transplantation to delete 1 allele of Nf1 in endothelial cells, vascular smooth muscle cells, and BMDCs alone, we determined which cell lineage is critical for neointima formation in vivo in mice. Here we demonstrate that heterozygous inactivation of Nf1 in BMDCs alone was necessary and sufficient for neointima formation after vascular injury and provide evidence of vascular inflammation in Nf1+/- mice. Further, analysis of peripheral blood from NF1 patients without overt vascular disease revealed increased concentrations of inflammatory cells and cytokines previously linked to vascular inflammation and vasoocclusive disease. These data provide genetic and cellular evidence of vascular inflammation in NF1 patients and Nf1+/- mice and provide a framework for understanding the pathogenesis of NF1 vasculopathy and potential therapeutic and diagnostic interventions.

Dual Lineage-Specific Expression of Sox17 During Mouse Embryogenesis

Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm‐derived organs are well known to originate from Sox17‐expressing cells, it is less certain whether fetal HSCs also originate from Sox17‐expressing cells. By generating a Sox17GFPCre allele and using it to assess the fate of Sox17‐expressing cells during embryogenesis, we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17‐positive cells. Prior to E9.5, the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para‐aortic splanchnopleural region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using fluorescence‐activated cell sorting together with RNA‐Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage‐specific marker. STEM Cells2012;30:2297–2308

The p85α Subunit of Class IA Phosphatidylinositol 3-Kinase Regulates the Expression of Multiple Genes Involved in Osteoclast Maturation and Migration

ABSTRACT Intracellular signals involved in the maturation and function of osteoclasts are poorly understood. Here, we demonstrate that osteoclasts express multiple regulatory subunits of class IA phosphatidylinositol 3-kinase (PI3-K) although the expression of the full-length form of p85α is most abundant. In vivo, deficiency of p85α results in a significantly greater number of trabeculae and significantly lower spacing between trabeculae as well as increased bone mass in both males and females compared to their sex-matched wild-type controls. Consistently, p85α−/− osteoclast progenitors show impaired growth and differentiation, which is associated with reduced activation of Akt and mitogen-activated protein kinase extracellular signal-regulated kinase 1 (Erk1)/Erk2 in vitro. Furthermore, a significant reduction in the ability of p85α−/− osteoclasts to adhere to as well as to migrate via integrin αvβ3 was observed, which was associated with reduced bone resorption. Microarray as well as quantitative real-time PCR analysis of p85α−/− osteoclasts revealed a significant reduction in the expression of several genes associated with the maturation and migration of osteoclasts, including microphathalmia-associated transcription factor, tartrate-resistant acid phosphatase, cathepsin K, and β3 integrin. Restoring the expression of the full-length form of p85α but not the version with a deletion of the Src homology-3 domain restored the maturation of p85α−/− osteoclasts to wild-type levels. These results highlight the importance of the full-length version of the p85α subunit of class IA PI3-K in controlling multiple aspects of osteoclast functions.

Essential role for focal adhesion kinase in regulating stress hematopoiesis

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been extensively studied in fibroblasts; however its function in hematopoiesis remains an enigma. FAK is thought to be expressed in myeloid and erythroid progenitors, and its expression is enhanced in response to cytokines such as granu-locyte macrophage colony-stimulating factor. Furthermore, bone marrow cells cultured in granulocyte macrophage colony-stimulating factor show active migration and chemoattractant-induced polarization, which correlates with FAK induction. While loss of FAK in mice results in embryonic lethality, we have deleted FAK in the adult bone marrow. We show an essential role for FAK in regulating hemolytic, myelotoxic, as well as acute inflammatory stress responses in vivo. In vitro, loss of FAK in erythroid and myeloid progenitors results in impaired cytokine induced growth and survival, as well as defects in the activation and expression of antiapoptotic proteins caspase 3 and Bcl-x(L). Additionally, reduced migration and adhesion of myeloid cells on extracellular matrix proteins, as well as impaired activation of Rac GTPase is also observed in the absence of FAK. Our studies reveal an essential role for FAK in integrating growth/survival and adhesion based functions in myeloid and erythroid cells predominantly under conditions of stress.

Functional B-1 progenitor cells are present in the hematopoietic stem cell-deficient embryo and depend on Cbfβ for their development.

Significance All lymphoid cells are considered to be products of hematopoietic stem cells (HSCs); however, it has been suggested, but not proven, that innate immune B-1 progenitor cells develop independently of HSCs in the fetal liver. B-1 cells, especially B-1a cells, are not replaced by adult bone marrow transplantation. Thus, it is critical to understand the origin and mechanisms required to sustain these cells in vivo because B-1 cells play important roles in the first line of defense against microbial infection and in preventing organ damage in autoimmune patients and infections in some patients after bone-marrow transplantation. We demonstrate that B-1 progenitor cells can develop independently of HSCs in the fetal liver and that their development relies critically on the expression of core-binding factor beta. The fetal liver is a major hematopoietic site containing progenitor cells that give rise to nearly all blood cells, including B-1 cells. Because the fetal liver is not a de novo site of hematopoietic stem cell (HSC) or progenitor-cell emergence, it must be seeded by yolk sac (YS)-derived erythromyeloid progenitors at embryonic day (E) 8.5–E10 and aorta-gonado-mesonephros (AGM)-derived HSCs at E10.5–E11.5. Although the B-1 progenitor cell pool in the fetal liver is considered to be of HSC origin, we have previously proposed that YS-derived B-1 progenitors may also contribute to this pool. Until now, it has been impossible to determine whether HSC-independent B-1 progenitor cells exist in the fetal liver. Here, we demonstrate the presence of transplantable fetal-liver B-1 and marginal zone B progenitor cells in genetically engineered HSC-deficient embryos. HSC-deficient YS and AGM tissues produce B-1 progenitors in vitro and thus may serve as sites of origin for the B-1 progenitors that seed the fetal liver. Furthermore, we have found that core-binding factor beta (Cbfβ) expression is required for fetal-liver B-1 progenitor cell maturation and expansion. Our data provide, to our knowledge, the first evidence for the presence of B-1 progenitor cells in the fetal liver that arise independently of HSCs and implicate Cbfβ as a critical molecule in the development of this lineage.