Gregor B. Adams

Osteoblastic cells regulate the haematopoietic stem cell niche

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by γ-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor

During mammalian ontogeny, haematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, where haematopoiesis occurs throughout adulthood. Unique features of bone that contribute to a microenvironmental niche for stem cells might include the known high concentration of calcium ions at the HSC-enriched endosteal surface. Cells respond to extracellular ionic calcium concentrations through the seven-transmembrane-spanning calcium-sensing receptor (CaR), which we identified as being expressed on HSCs. Here we show that, through the CaR, the simple ionic mineral content of the niche may dictate the preferential localization of adult mammalian haematopoiesis in bone. Antenatal mice deficient in CaR had primitive haematopoietic cells in the circulation and spleen, whereas few were found in bone marrow. CaR-/- HSCs from fetal liver were normal in number, in proliferative and differentiative function, and in migration and homing to the bone marrow. Yet they were highly defective in localizing anatomically to the endosteal niche, behaviour that correlated with defective adhesion to the extracellular matrix protein, collagen I. CaR has a function in retaining HSCs in close physical proximity to the endosteal surface and the regulatory niche components associated with it.

Oocyte Generation in Adult Mammalian Ovaries by Putative Germ Cells in Bone Marrow and Peripheral Blood

It has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood.

Therapeutic targeting of a stem cell niche

The specialized microenvironment or niche where stem cells reside provides regulatory input governing stem cell function. We tested the hypothesis that targeting the niche might improve stem cell–based therapies using three mouse models that are relevant to clinical uses of hematopoietic stem (HS) cells. We and others previously identified the osteoblast as a component of the adult HS cell niche and established that activation of the parathyroid hormone (PTH) receptor on osteoblasts increases stem cell number. Here we show that pharmacologic use of PTH increases the number of HS cells mobilized into the peripheral blood for stem cell harvests, protects stem cells from repeated exposure to cytotoxic chemotherapy and expands stem cells in transplant recipients. These data provide evidence that the niche may be an attractive target for drug-based stem cell therapeutics.

Hematopoietic stem cells depend upon Gsα-mediated signalling to engraft bone marrow

Haematopoietic stem and progenitor cells (HSPCs) change location during development and circulate in mammals throughout life, moving into and out of the bloodstream to engage bone marrow niches in sequential steps of homing, engraftment and retention. Here we show that HSPC engraftment of bone marrow in fetal development is dependent on the guanine-nucleotide-binding protein stimulatory α subunit (Gαs). HSPCs from adult mice deficient in Gαs (Gαs-/-) differentiate and undergo chemotaxis, but also do not home to or engraft in the bone marrow in adult mice and demonstrate a marked inability to engage the marrow microvasculature. If deleted after engraftment, Gαs deficiency did not lead to lack of retention in the marrow, rather cytokine-induced mobilization into the blood was impaired. Testing whether activation of Gαs affects HSPCs, pharmacological activators enhanced homing and engraftment in vivo. Gαs governs specific aspects of HSPC localization under physiological conditions in vivo and may be pharmacologically targeted to improve transplantation efficiency.

Nf2/Merlin Regulates Hematopoietic Stem Cell Behavior by Altering Microenvironmental Architecture

Stem cell population size is highly regulated across species and tissue types, and alterations are associated with premature tissue failure or cancer. We assessed whether the tumor suppressor and mediator of cell contact inhibition Nf2/merlin plays a role in governing the hematopoietic stem cell pool by stem cell-autonomous or niche-determined processes. Hematopoietic stem cells in Nf2-deficient mice were increased in number and demonstrated a marked shift in location to the circulation. These changes were entirely dependent on changes in the microenvironment, with a marked increase in trabecular bone and marrow vascularity associated with increased VEGF, but without cell-autonomous alterations in stem cell characteristics. Nf2/merlin is critical for maintaining normal structure and function of the hematopoietic stem cell niche. It limits both bone and vascular components, and our model suggests that it thereby constrains stem cell number and position.

Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat.

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4+ cell count <400 cells/mm3). We examined the efficiency of transduction and transgene expression in adult bone marrow (BM)- and umbilical cord blood (UCB)-derived CD34+ cells induced to differentiate into T cells and monocytes in vitro with an MuLV-based vector encoding the neomycin resistance gene and an intracellular antibody directed against the Tat protein of HIV-1 (sFvtat1-Ckappa). The expression of the marker gene and the effects of antiviral construct on subsequent challenge with monocytotropic and T cell-tropic HIV-1 isolates were monitored in vitro in purified T cells and monocytes generated in culture from the transduced CD34+ cells. Transduction efficiencies of CD34+ cells ranged between 22 and 27%. Differentiation of CD34+ cells into T cells or monocytes was not significantly altered by the transduction process. HIV-1 replication in monocytes and CD4+ T cells derived from CD34+ cells transduced with the intracellular antibody gene was significantly reduced in comparison with the degree of HIV replication seen in monocytes and CD4+ T cells derived from CD34+ cells transduced with the neomycin resistance gene alone. Further, T cells and monocytes derived from CD34+ cells transduced with the intracellular antibody gene were demonstrated to express the sFvtat1-Ckappa transgene by RT-PCR and had a selective growth advantage in cultures that had been challenged with HIV-1. These data demonstrate that sFvtat1-Ckappa inhibits HIV-1 replication in T cells and monocytes developing from CD34+ cells and supports the continuing development of a stem cell gene therapy for the treatment of HIV-1 infection.

The in vivo effects of combination antiretroviral drug therapy on peripheral blood CD34+ cell colony-forming units from HIV type 1-infected patients

This study investigated the effects of a combination antiretroviral drug regimen (indinavir and two nucleoside analogs or ritonavir and saquinavir) on the levels of CD34+ colony-forming units (CFU-Cs) in the peripheral blood of HIV-1+ patients. Ten patients who were receiving combination antiretroviral drug therapy were studied and their peripheral blood CD34+ CFU-Cs were measured prior to, 1 month after, and 4 to 6 months after the commencement of therapy. The levels of CD4+ T cells increased significantly in these patients (paired t test, p = 0.0027) and plasma viral load became undetectable in all but one patient studied. Measurements of the CFU-Cs showed that their levels tended to increase on the commencement of therapy, and these levels became significantly higher than baseline by 4-6 months (paired t test, p = 0.0293). Analysis of the different colony phenotype demonstrated that the main contributor to this increase consisted of burst-forming unit erythroid (BFU-E) cells. These data also demonstrated that there was an inverse correlation between the rise in CFU-Cs at 4-6 months compared with CD4+ cell, CD8+ cell, and neutrophil counts, and hemoglobin concentration, at baseline. The demonstrated increase in the levels of CD34+ CFU-Cs suggests that HIV-1 may have an inhibitory effect on these cells in vivo, and that this inhibition may be abrogated by suppression of viral replication.

Tissue source dictates lineage outcome of human fetal CD34 CD38 cells

OBJECTIVE The translocation from fetal liver hematopoiesis to secondary organs occurs during the second trimester of human gestation. It has been hypothesized that stem cells migrate and acquire lineage potential based on cues specific to the adopted microenvironment. We evaluated primitive hematopoietic cell populations in the fetal human to determine if lineage restriction precedes or follows translocation to sites of hematopoietic activity including thymus, spleen, bone marrow, and liver. METHODS Sets of hematopoietic tissues from individual second-trimester human abortuses were used to compare and quantitate the lineage outcome of immunophenotypically primitive cells from each of the hematopoietic organs using ex vivo myeloid and lymphoid differentiation systems. RESULTS Despite uniformity in immunophenotype, functional capabilities were highly restricted by the tissue of origin and alteration in the ex vivo differentiation context did not lead to a change in differentiation outcome. CONCLUSION Translocation of primitive cells from fetal liver to tissues of mature hematopoietic activity is associated with tissue-specific, quantitative changes in differentiation potential that are unresponsive to alternative differentiation environments. These data suggest that multipotentiality is lost prior to or upon stem-cell migration in the developing human. It is not persistent with residence in a secondary hematopoietic organ.

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts

BACKGROUND Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose. METHODS MFGnlslacZ retroviral vector, packaged in high-titer, split-genome packaging cell line (FLYA4) was used to transduce the skeletal myoblast cell line L6. L6 cells, cultured in 10% fetal calf serum, were transduced with the MFGnlslacZ vector by means of filtered supernatant from FLYA4 cells. Transduced L6 cells were divided into four groups. Group I cells were fixed as myoblasts 3 days after transduction. Group II cells were allowed to differentiate into myotubes. Group III cells were split every 3 days for 4 months. Group IV cells were split as in group III but then allowed to differentiate into myotubes. All samples were fixed and stained for beta-galactosidase activity. The effects on gene transfer of transforming growth factor-beta, insulin-like growth factor-I, and platelet-derived growth factor were determined by spectrophotometric assay of beta-galactosidase activity in cells transduced in the presence or absence of serum with 0 to 200 ng/ml of each growth factor. RESULTS Morphometric analysis showed that 66.3% +/- 3% to 69.6% +/- 6% of cells in group I to IV expressed the lacZ reporter gene. In the presence of serum, transforming growth factor-beta significantly inhibited gene transfer, whereas insulin-like growth factor-I and platelet-derived growth factor significantly enhanced gene transfer. In absence of serum, however, only platelet-derived growth factor enhanced retrovirally mediated gene transfer into skeletal myoblasts. CONCLUSION MFG retroviral vectors packaged in FLYA4 cells are efficient in gene transfer into skeletal myoblasts and result in transgenic expression that is maintained after repeated cell division, differentiation, or both. Platelet-derived growth factor enhances retrovirally mediated gene transfer into skeletal myoblasts.