R4 RGS proteins suppress engraftment of human hematopoietic stem/progenitor cells by modulating SDF-1/CXCR4 signaling.

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM) microenvironment are tightly regulated by the chemokine stromal cell-derived factor-1 (SDF-1) and its G-protein-coupled receptor C-X-C motif chemokine receptor 4 (CXCR4), which on engagement with G-protein subunits, trigger downstream migratory signals. Regulators of G-protein signaling (RGS) are GTPase-accelerating protein of the Gα subunit and R4 subfamily members have been implicated in SDF-1-directed trafficking of mature hematopoietic cells, yet their expression and influence on HSPCs remain mostly unknown. Here, we demonstrated that human CD34+ cells expressed multiple R4 RGS genes, of which RGS1, RGS2, RGS13, and RGS16 were significantly upregulated by SDF-1 in a CXCR4-dependent fashion. Forced overexpression of RGS1, RGS13, or RGS16 in CD34+ cells not only inhibited SDF-1-directed migration, calcium mobilization, and phosphorylation of AKT, ERK, and STAT3 in vitro, but also markedly reduced BM engraftment in transplanted NOD/SCID mice. Genome-wide microarray analysis of RGS-overexpressing CD34+ cells detected downregulation of multiple effectors with established roles in stem cell trafficking/maintenance. Convincingly, gain-of-function of selected effectors or ex vivo priming with their ligands significantly enhanced HSPC engraftment. We also constructed an evidence-based network illustrating the overlapping mechanisms of RGS1, RGS13, and RGS16 downstream of SDF-1/CXCR4 and Gαi. This model shows that these RGS members mediate compromised kinase signaling and negative regulation of stem cell functions, complement activation, proteolysis, and cell migration. Collectively, this study uncovers an essential inhibitory role of specific R4 RGS proteins in stem cell engraftment, which could potentially be exploited to develop improved clinical HSPC transplantation protocols.

Ubiquitous expression of MAKORIN-2 in normal and malignant hematopoietic cells and its growth promoting activity.

Makorin-2 (MKRN2) is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM) compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.

Generation of NSE-MerCreMer transgenic mice with tamoxifen inducible Cre activity in neurons.

To establish a genetic tool for conditional deletion or expression of gene in neurons in a temporally controlled manner, we generated a transgenic mouse (NSE-MerCreMer), which expressed a tamoxifen inducible type of Cre recombinase specifically in neurons. The tamoxifen inducible Cre recombinase (MerCreMer) is a fusion protein containing Cre recombinase with two modified estrogen receptor ligand binding domains at both ends, and is driven by the neural-specific rat neural specific enolase (NSE) promoter. A total of two transgenic lines were established, and expression of MerCreMer in neurons of the central and enteric nervous systems was confirmed. Transcript of MerCreMer was detected in several non-neural tissues such as heart, liver, and kidney in these lines. In the background of the Cre reporter mouse strain Rosa26R, Cre recombinase activity was inducible in neurons of adult NSE-MerCreMer mice treated with tamoxifen by intragastric gavage, but not in those fed with corn oil only. We conclude that NSE-MerCreMer lines will be useful for studying gene functions in neurons for the conditions that Cre-mediated recombination resulting in embryonic lethality, which precludes investigation of gene functions in neurons through later stages of development and in adult.

Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, ß-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

3' Splicing variants of ret receptor tyrosine kinase are differentially expressed in mouse embryos and in adult mice.

The RET protooncogene encodes for a transmembrane receptor tyrosine kinase and plays a crucial role in nephrogenesis and the enteric nervous system (ENS) development. Alternative splicing at the 3' end of the RET gene generates 3' splicing variants that encode RET 9, RET 51 and RET 43 isoforms. It has been hypothesized that these isoforms perform distinct functions and that their expressions are differentially regulated during mammalian development. To gain an insight into the expression patterns of various ret isoforms during embryogenesis, we investigate the temporal and spatial expressions of ret gene in mouse embryos and in adult mice. We characterized the 3' end of the mouse ret gene and localized the alternatively spliced exons. Using 3' rapid amplification of cDNA ends (3' RACE) and reverse transcription-polymerase chain reaction (RT-PCR), ret 9 and ret 51 transcripts were identified in both mouse embryos and adult mouse tissues. However, the ret 43 transcript was not. Using in situ hybridization, we showed that ret 9 was the dominant ret encoding transcript in mouse embryos. Transcripts of ret 9 were detected in all cranial ganglia; in the sensory and autonomic ganglia of the trunk; in a subset of neurons of the dorsal root ganglion (DRG); in the motor neurons of the spinal cord; in the developing lung and excretory systems; in the enteric neuroblasts of the ENS; and in the thyroid lobes. In contrast, ret 51 expression was weak and restricted to the motor column of the spinal cord, the DRG, the enteric neuroblasts, the lung bud and the kidney. In adult mice, ret 9 expression was relatively widespread in many organs while that of ret 51 was rather restricted. Our data indicated that ret isoforms are temporally and spatially regulated in mouse embryos and adult mouse.