Forward RNAi screens in primary human hematopoietic stem/progenitor cells.

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.

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.

Endoglin is not critical for hematopoietic stem cell engraftment and reconstitution but regulates adult erythroid development.

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract.

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.

A road map toward defining the role of Smad signaling in hematopoietic stem cells.

The transforming growth factor-beta (TGF-beta) superfamily encompasses the ligands and receptors for TGF-beta, bone morphogenic proteins (BMPs), and Activins. Cellular response to ligand is context-dependent and may be controlled by specificity and/or redundancy of expression of these superfamily members. Several pathways within this family have been implicated in the proliferation, differentiation, and renewal of hematopoietic stem cells (HSCs); however, their roles and redundancies at the molecular level are poorly understood in the rare HSC. Here we have characterized the expression of TGF-beta superfamily ligands, receptors, and Smads in murine HSCs and in the Lhx2-hematopoietic progenitor cell (Lhx2-HPC) line. We demonstrate a remarkable likeness between these two cell types with regard to expression of the majority of receptors and Smads necessary for the transduction of signals from TGF-beta, BMP, and Activin. We have also evaluated the response of these two cell types to various ligands in proliferation assays. In this regard, primary cells and the Lhx2-HPC line behave similarly, revealing a suppressive effect of Activin-A that is similar to that of TGF-beta in bulk cultures and no effect of BMP-4 on proliferation. Signaling studies that verify the phosphorylation of Smad2 (Activin and TGF-beta) and Smad1/5 (BMP) confirm cytosolic responses to these ligands. In addition to providing a thorough characterization of TGF-beta superfamily expression in HSCs, our results define the Lhx2-HPC line as an appropriate model for molecular characterization of Smad signaling.