Ramesh Nayak

Rho GTPases control specific cytoskeleton-dependent functions of hematopoietic stem cells

The Rho family of guanosine triphosphatases (GTPases) is composed of members of the Ras superfamily of proteins. They are GTP‐bound molecules with a modest intrinsic GTPase activity that can be accelerated upon activation/localization of specialized guanine nucleotide exchange factors. Members of this family act as molecular switches and are required for coordinated cytoskeletal rearrangements that are crucial in a set of specialized functions of mammalian stem cells. These functions include self‐renewal, adhesion, and migration. Mouse gene‐targeting studies have provided convincing evidence of the indispensable and dispensable roles of individual members of the Rho GTPase family and the putative upstream and downstream mediators in stem cell‐specific functions. The role of Rho GTPases and related signaling pathways previously seen in other cell types and organisms have been confirmed in mammalian hematopoietic stem cells (HSCs), and new signaling pathways and unexpected functions unique to HSCs have been identified and dissected. This review summarizes our current understanding of the role of Rho family of GTPases on HSC and progenitor activity through cytoskeleton‐mediated signaling pathways, providing insight about relevant signaling pathways that regulate mammalian stem cell self‐renewal, adhesion, and migration.

Klf5 controls bone marrow homing of stem cells and progenitors through Rab5-mediated β1/β2-integrin trafficking

Kruppel-like factor 5 (Klf5) regulates pluripotent stem cell self-renewal but its role in somatic stem cells is unknown. Here we show that Klf5 deficient haematopoietic stem cells and progenitors (HSC/P) fail to engraft after transplantation. This HSC/P defect is associated with impaired bone marrow homing and lodging and decreased retention in bone marrow, and with decreased adhesion to fibronectin and expression of membrane-bound β1/β2-integrins. In vivo inducible gain-of-function of Klf5 in HSCs increases HSC/P adhesion. The expression of Rab5 family members, mediators of β1/β2-integrin recycling in the early endosome, is decreased in Klf5Δ/Δ HSC/Ps. Klf5 binds directly to the promoter of Rab5a/b and overexpression of Rab5b rescues the expression of activated β1/β2-integrins, adhesion and bone marrow homing of Klf5Δ/Δ HSC/Ps. Altogether, these data indicate that Klf5 is indispensable for adhesion, homing, lodging and retention of HSC/Ps in the bone marrow through Rab5-dependent post-translational regulation of β1/β2 integrins.

Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells

Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN patient-derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues the dysgranulopoietic defect in SCN patient-derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to correct NE mislocalization.

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.

Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

Vasculopathy-associated hyperangiotensinemia mobilizes haematopoietic stem cells/progenitors through endothelial AT₂R and cytoskeletal dysregulation.

Patients in organ failure of vascular origin have increased circulating hematopoietic stem cells and progenitors (HSC/P). Plasma levels of angiotensin II (Ang-II), are commonly increased in vasculopathies. Hyperangiotensinemia results in activation of a very distinct Ang-II receptor set, Rho-family GTPase members, and actin in bone marrow endothelial cells (BMEC) and HSC/P, which results in decreased membrane integrin activation in both BMEC and HSC/P, and in HSC/P de-adhesion and mobilization. The Ang-II effect can be reversed pharmacologically and genetically by inhibiting Ang-II production or signaling through BMEC AT2R, HSCP AT1R/AT2R or HSC/P RhoA, but not by interfering with other vascular tone mediators. Hyperangiotensinemia and high counts of circulating HSC/P seen in sickle cell disease (SCD) as a result of vascular damage, is significantly decreased by Ang-II inhibitors. Our data define for the first time the role of Ang-II HSC/P traffic regulation and redefine the hematopoietic consequences of anti-angiotensin therapy in SCD.

p190-B RhoGAP regulates the functional composition of the mesenchymal microenvironment.

Hematopoiesis is regulated by components of the microenvironment, so-called niche. Here, we show that p190-B GTPase-activating protein (p190-B) deletion in mice causes hematopoietic failure during ontogeny, in p190-B−/− fetal liver and bones, and in p190-B+/− adult bones and spleen. These defects are non-cell autonomous, as we previously showed that transplantation of p190-B−/− hematopoietic cells into wild-type (WT) hosts leads to normal hematopoiesis. Coculture of mesenchymal stem (MSC)/progenitor cells and wild-type bone marrow (BM) cells reveals that p190-B−/− MSCs are dysfunctional in supporting hematopoiesis owing to impaired Wnt signaling. Furthermore, p190-B loss causes alteration in BM niche composition, including abnormal colony-forming unit (CFU)-fibroblast, CFU-adipocyte and CFU-osteoblast numbers. This is due to altered MSC lineage fate specification to osteoblast and adipocyte lineages. Thus, p190-B organizes a functional mesenchymal/microenvironment for normal hematopoiesis during development.

444. Large Scale Culture and Differentiation of Induced Pluripotent Stem Cells for Neutrophil Replacement Therapies

Neutrophils are a key component in the innate immune system and are crucial in the protection against bacterial and fungal infections. Patients with neutropenias are at high risk to develop serious life threatening bacterial and fungal infections. Transfusion of donor neutrophils into neutropenic patients may help alleviate disease burden, but difficulty in collecting and transfusing sufficient quantities of viable donor neutrophils has limited the clinical adaptation of neutrophil replacement therapies. Induced pluripotent stem cells (iPSC) are an attractive alternative source to donor derived neutrophils due to their ability to self-renew and differentiate into cells of the three embryonic germ layers, including neutrophils. Successful implementation of iPSC for neutrophil replacement therapies will require a) derivation of iPSC, b) large scale culture of iPSC, and c) efficient differentiation of these iPSC into functional neutrophils in xeno-fee, GMP-compliant conditions. First, normal human fibroblasts were reprogrammed in xeno-free, GMP compliant conditions though nucleofection of a non-integrating 3 plasmid system carrying OCT4, SOX2, KLF-4, l-MYC, Lin28 and shp53. The reprogrammed fibroblasts were cultured in defined conditions on Vitronectin XF in TeSR-E7 media until colony formation when they were transitioned to E8 medium. We generated 3 lines under these conditions and all iPSC derived and cultured under these conditions retain a normal karyotype, express pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 at >85% as determined by FACs analysis, and differentiate into all three germ layers in vitro. Once iPSC lines were established, cultures were adapted from adherent cultures to non-adherent, aggregate suspension cultures in spinner flask bioreactor systems in mTeSR or E8 medium. Briefly, adherent iPSC are dissociated into single cells and seeded into a spinner flask at a concentration of 2×105 cells/ml and spun at 60 RPM. We evaluated 2 lines in 3 independent experiments and cells readily formed clusters and were able to achieve 4-log expansion (range: 2-4 log) within approximately 40 days (10 passages). All lines retained a normal karyotype, expressed pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 (>85%), and displayed a normal morphology when re-plated in adherent culture. We next adapted a neutrophil differentiation system for differentiation from iPSC from the defined media/spinnerflask system. Clusters of cells from spinner flask cultures were induced to form embryoid bodies (EBs) by transitioning the cultures to EB specification medium for 5 days, followed by a neutrophil specification medium containing IL-3 and G-CSF in adherent conditions. After 7-14 days in neutrophil specification conditions, EBs attached to the plate and CD45+CD11b+CD16+ neutrophils were shed into the medium and were harvested twice weekly for 30-60 days. In 10 experiments, we collected an average of 6.5×10^5 CD45+ cells per well of differentiation representing a 6.5 fold expansion (range: 3-10 fold). In conclusion, iPSC have been derived in GMP compliant conditions and can be cultured at a large scale using spinner flask bioreactors, and the iPSC from spinner flask bioreactors can then successfully be differentiated into CD45+ cells. Thus, iPSC represent an attractive, self-renewing resource of neutrophils for neutrophil replacement therapies.

Regulation of the Cytoskeleton by the Rho Family of GTPases in Hematopoietic Stem Cells in Health and Disease

The Rho family of GTPases is composed of members of the Ras superfamily of proteins. They are GTP-bound molecules with a modest intrinsic GTPase activity that can be accelerated upon activation/localization of specialized guanine nucleotide exchange factors. Members of this family act as molecular switches, and are required for coordinated cytoskeletal rearrangements which are crucial in a set of specialized functions of mammalian stem cells. These functions include self-renewal, adhesion, and migration. Mouse gene-targeting studies have provided convincing evidence of the indispensable and dispensable roles of individual members of the Rho GTPase family, and the putative upstream and downstream mediators in stem cell-specific functions. The role of Rho GTPases and related signaling pathways previously seen in other cell types and organisms have been confirmed in mammalian hematopoietic stem cells (HSC), and new signaling pathways and unexpected functions unique to HSC have been identified and dissected. This review intends to summarize our current understanding of the role of the Rho family of GTPases on HSC and progenitor activity through cytoskeleton-mediated signaling pathways, and to provide insight on relevant signaling pathways that regulate mammalian stem cell self-renewal, adhesion and migration in health and HSC disease.