Regulation of Blood Stem Cell Development.

Understanding how the blood system is formed is an ongoing fundamental research challenge. Developmental biology has provided many insights into the molecules and processes that affect the formation of the blood tissues, both in health and disease. It is of particular interest for clinical transplantation therapies to understand how hematopoietic stem cells (HSCs)-the self-renewing purveyors of the adult blood system that produce over 10 different functionally specialized cell lineages and over 10(11) cells daily-are generated during embryonic stages. Recent successes to reprogram the fate of adult differentiated cells to pluripotency and to other cell lineages now highlight the importance of identifying the cells and molecules that affect the in vivo developmental initiation of rare and robust transplantable HSCs. The close association of the developing hematopoietic and vascular system, hematopoietic cell mobility through the circulation, and the essential role of the embryonic hematopoietic system in adult hematopoietic cell development make this a formidable study. This chapter reviews the advances, controversies, and current state of our knowledge of the growing field of hematopoietic development, with a special focus on the regulation of the natural transdifferentiation of endothelial cells to HSCs within the developing embryo.

The biochemistry of hematopoietic stem cell development.

BACKGROUND: The cornerstone of the adult hematopoietic system and clinical treatments for blood-related disease is the cohort of hematopoietic stem cells (HSC) that is harbored in the adult bone marrow microenvironment. Interestingly, this cohort of HSCs is generated only during a short window of developmental time. In mammalian embryos, hematopoietic progenitor and HSC generation occurs within several extra- and intraembryonic microenvironments, most notably from 'hemogenic' endothelial cells lining the major vasculature. HSCs are made through a remarkable transdifferentiation of endothelial cells to a hematopoietic fate that is long-lived and self-renewable. Recent studies are beginning to provide an understanding of the biochemical signaling pathways and transcription factors/complexes that promote their generation. SCOPE OF REVIEW: The focus of this review is on the biochemistry behind the generation of these potent long-lived self-renewing stem cells of the blood system. Both the intrinsic (master transcription factors) and extrinsic regulators (morphogens and growth factors) that affect the generation, maintenance and expansion of HSCs in the embryo will be discussed. MAJOR CONCLUSIONS: The generation of HSCs is a stepwise process involving many developmental signaling pathways, morphogens and cytokines. Pivotal hematopoietic transcription factors are required for their generation. Interestingly, whereas these factors are necessary for HSC generation, their expression in adult bone marrow HSCs is oftentimes not required. Thus, the biochemistry and molecular regulation of HSC development in the embryo are overlapping, but differ significantly from the regulation of HSCs in the adult. GENERAL SIGNIFICANCE: HSC numbers for clinical use are limiting, and despite much research into the molecular basis of HSC regulation in the adult bone marrow, no panel of growth factors, interleukins and/or morphogens has been found to sufficiently increase the number of these important stem cells. An understanding of the biochemistry of HSC generation in the developing embryo provides important new knowledge on how these complex stem cells are made, sustained and expanded in the embryo to give rise to the complete adult hematopoietic system, thus stimulating novel strategies for producing increased numbers of clinically useful HSCs. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Stem cell self-renewal: lessons from bone marrow, gut and iPS toward clinical applications.

The hematopoietic stem cell (HSC) is the prototype organ-regenerating stem cell (SC), and by far the most studied type of SC in the body. Currently, HSC-based therapy is the only routinely used SC therapy; however, advances in the field of embryonic SCs and induced pluripotent SCs may change this situation. Interest into in vitro generation of HSCs, including signals for HSC expansion and differentiation from these more primitive SCs, as well as advances in other organ-specific SCs, in particular the intestine, provide promising new applications for SC therapies. Here, we review the basic principles of different SC systems, and on the basis of the experience with HSC-based SC therapy, provide recommendations for clinical application of emerging SC technologies.

Expression analysis of the TAB2 protein in adult mouse tissues.

BACKGROUND: The Interleukin-1 (IL-1) signaling component TAK1 binding protein 2 (TAB2) plays a role in activating the NFkappaB and JNK signaling pathways. Additionally, TAB2 functions in the nucleus as a repressor of NFkappaB-mediated gene regulation. OBJECTIVE: To obtain insight into the function of TAB2 in the adult mouse, we analyzed the in vivo TAB2 expression pattern. MATERIALS AND METHODS: Cell lines and adult mouse tissues were analyzed for TAB2 protein expression and localization. RESULTS: Immunohistochemical staining for TAB2 protein revealed expression in the vascular endothelium of most tissues, hematopoietic cells and brain cells. While TAB2 is localized in both the nucleus and the cytoplasm in cell lines, cytoplasmic localization predominates in hematopoietic tissues in vivo. CONCLUSIONS: The TAB2 expression pattern shows striking similarities with previously reported IL-1 receptor expression and NFkappaB activation patterns, suggesting that TAB2 in vivo is playing a role in these signaling pathways.

Developmental origins of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are at the foundation of the hematopoietic hierarchy and give rise to all blood lineages in the adult organism. A thorough understanding of the molecular, cellular, and developmental biology of HSCs is of fundamental importance, but is also clinically relevant for the advancement of cell replacement therapies and transplantation protocols in blood-related genetic disease and leukemias. While the major anatomical sites of hematopoiesis change during ontogeny, it was long believed that the developmental origin of the adult mammalian hematopoietic system was the yolk sac. However, current studies have shown that the first adult-type HSCs are autonomously generated in the intrabody portion of the mouse embryo, the aorta-gonads-mesonephros (AGM) region, and sublocalize to the dorsal aorta. HSCs are also found in the other large embryonic vessels, the vitelline and umbilical arteries. The intraluminal hematopoietic clusters along these vessels, together with the role of the Runx1 transcription factor in cluster and HSC formation and the HSC/endothelial/mesenchymal Runxl expression pattern, strongly suggest a vascular endothelial/mesenchymal origin for the first HSCs. Moreover, a transgenic mouse line expressing the GFP marker under the control of the Sca-1 transcriptional regulatory elements (GFP expression marks all HSCs) shows a clear localization of GFP-expressing cells to the endothelial cell layer of the dorsal aorta. Thus, highly enriched GFP-positive AGM HSCs will serve as a basis for the future examination of the cellular and molecular factors involved in the induction and expansion of adult HSCs.

Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week6) in extended long-term cultures.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta-gonad-mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34(+) umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC(week6) produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

Cloning of the Ly-6A (Sca-1) gene locus and identification of a 3' distal fragment responsible for high-level gamma-interferon-induced expression in vitro.

The Ly-6A and Ly-6E allelic genes encode the Sca-1 protein, which is one of the most widely used markers in haematopoietic stem cell isolation procedures. Identification of the specific gene regulatory elements that direct haematopoietic stem cell specific expression of Sca-1 is of current interest for purposes of stem cell manipulation. Both the Ly-6E and Ly-6A alleles have been examined for regions containing DNase I hypersensitive sites thought to be indicative of transcriptional regulatory elements. In these previous studies, the Ly-6E allele with its flanking regulatory sequences was cloned, and the region responsible for high-level gamma-interferon (gamma-IFN)-induced expression was localized to a 3' distal sequence containing two strong DNase1 hypersensitive sites. Because the Ly-6A allele is thought to provide higher levels of expression in haematopoietic stem cells, we isolated over 25 kb of the Ly-6A gene and flanking regulatory regions. We show here that sequences analogous to those in the Ly-6E allele are responsible for high-level gamma-IFN-induced expression in vitro. Furthermore, we show that this 3' distal Ly-6A fragment directs high-level gamma-IFN-induced expression from a heterologous promoter, suggesting that it is a potent enhancer that could be useful for expression in haematopoietic stem cells.

Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.

The AML1:CBFbeta transcription factor complex is essential for definitive hematopoiesis. Null mutations in mouse AML1 result in midgestational lethality with a complete lack of fetal liver hematopoiesis. While the cell autonomous nature and expression pattern of AML1 suggest an intrinsic role for this transcription factor in the developing hematopoietic system, no direct link to a functional cell type has been made. Here, we examine the consequences of AML1 loss in hematopoietic stem cells (HSC) of the mouse embryo. We demonstrate an absolute requirement for AML1 in functional HSCs. Moreover, haploinsufficiency results in a dramatic change in the temporal and spatial distribution of HSCs, leading to their early appearance in the normal position in the aorta-gonad-mesonephros region and also in the yolk sac.

CFU-S(11) activity does not localize solely with the aorta in the aorta-gonad-mesonephros region.

The aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site in the midgestation mouse conceptus and first contains colony-forming units-spleen day 11 (CFU-S(11)) at embryonic day 10 (E10). Because CFU-S(11) activity is present in the AGM region before the onset of hematopoietic stem cell (HSC) activity, CFU-S(11) activity in the complex developing vascular and urogenital regions of the AGM was localized. From E10 onward, CFU-S(11) activity is associated with the aortic vasculature, and is found also in the urogenital ridges (UGRs). Together with data obtained from organ explant cultures, in which up to a 16-fold increase in CFU-S(11) activity was observed, it was determined that CFU-S(11) can be increased autonomously both in vascular sites and in UGRs. Furthermore, CFU-S(11) activity is present in vitelline and umbilical vessels. This, together with the presence of CFU-S(11) in the UGRs 2 days before HSC activity, suggests both temporally and spatially distinct emergent sources of CFU-S(11). (Blood. 2000;96:2902-2904)

Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.

The aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site within the mammalian embryo body, and the first place from which hematopoietic stem cells (HSCs) emerge. Within the complex embryonic vascular, excretory and reproductive tissues of the AGM region, the precise location of HSC development is unknown. To determine where HSCs develop, we subdissected the AGM into aorta and urogenital ridge segments and transplanted the cells into irradiated adult recipients. We demonstrate that HSCs first appear in the dorsal aorta area. Furthermore, we show that vitelline and umbilical arteries contain high frequencies of HSCs coincident with HSC appearance in the AGM. While later in development and after organ explant culture we find HSCs in the urogenital ridges, our results strongly suggest that the major arteries of the embryo are the most important sites from which definitive HSCs first emerge.

Embryonic beginnings of definitive hematopoietic stem cells.

The ability of the many cell types within the adult blood system to be constantly replenished and renewed from hematopoietic stem cells is an interesting problem in development and differentiation and has led to questions concerning how, when and where these stem cells for the adult hematopoietic system are generated within the embryo. During embryonic development many mature hematopoietic cells appear before adult-type hematopoietic stem cells thus the notion of a conventional hematopoietic hierarchy is challenged. Experiments probing the development of hematopoietic stem cells in the mouse embryo strongly suggest that at least two independent hematopoietic sites generate blood cells during development; the yolk sac, which produces the transient embryonic hematopoietic system, and the AGM (aorta-gonad-mesonephros) region, which initiates the long-lived adult hematopoietic system.

Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins.

Strategies to inhibit the spread of HIV infection consist of a number of specific molecular approaches. Since viral production is dependent upon Tat-mediated transactivation of the HIV promoter through the Tat activating region (TAR), tat antisense RNA, anti-tat ribozymes, TAR decoys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant proteins, including a newly generated form Tat delta 58, for the ability to inhibit Tat-mediated transactivation and HIV production. In addition, we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (Tat delta 53) and the Drosophila Engrailed (Eng) transcription repressor domain to test the hypothesis that transcriptional repression can be targeted to the HIV promoter. This fusion mutant was also examined for its capacity to block both Tat-mediated transactivation and HIV replication. We show that three mutants Tat delta 53. Tat delta 58 and Tat delta 53/Eng result in a transdominant phenotype inhibiting wild-type Tat-mediated transactivation, and that the inhibiting potential is increased by the presence of the entire basic domain or the fusion of a repressor domain. However, only the transdominant mutants Tat delta 58 and Tat delta 53/Eng significantly inhibit HIV-1 replication after infection of transfected T cell lines. These results demonstrate the potent inhibiting activity of Tat mutants on HIV replication, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.

Development of the definitive hematopoietic hierarchy in the mouse.

Recent research on the ontogeny of the hematopoietic system in mammals has shown that a simple textbook steady-state hematopoietic hierarchy can not be strictly applied to the hematopoietic cells found within the embryo. During embryonic development, hematopoietic cells originate, migrate and differentiate in a number of distinct anatomical sites such as the yolk sac AGM region and liver and thus represent various classes of cells within diverse microenvironments. In this manuscript we review both cellular and molecular aspects of developmental hematopoiesis and present our current views on the numerous complex mechanisms underlying the establishment of definitive hematopoiesis.

High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.

Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV.

Expression of the Ly-6E.1 (Sca-1) transgene in adult hematopoietic stem cells and the developing mouse embryo.

The mouse hematopoietic marker Sca-1, encoded by the Ly-6E.1 and Ly-6A.2 genes, has been instrumental in the enrichment and characterization of the stem cell for the adult blood system. In the studies reported here, we use Ly-6E.1 genomic fragments to direct expression of a lacZ marker transgene in vivo to study Ly-6E.1 specific regulatory elements in the hematopoietic stem cell and to localize these cells in the developing mouse embryo. We demonstrate that a region approximately 9 kb downstream from the transcriptional start site is required for the distinct, restricted expression pattern of the Ly-6E.1-lacZ transgene within adult hematopoietic stem cells and embryos. We also demonstrate that viable and functional lacZ-expressing hematopoietic stem cells can be enriched by FDG staining and flow cytometric sorting. The Ly-6E.1-lacZ-mediated enrichment of hematopoietic stem cells from adult transgenic bone marrow in combination with the temporal expression pattern of the transgene in the pro/mesonephros suggest an intraembryonic site of development for these cells in the mouse.

HIV- I Nef severely impairs thymocyte development and peripheral T-cell function by a CD4-independent mechanism.

Nef is a regulatory protein of the human and simian immunodeficiency viruses (HIV and SIV) whose role in infection and the viral life cycle are not fully understood. In T-lymphocytes Nef down-regulates cell-surface CD4, and has been implicated in an increase in infectivity at low primary viral isolate titres. Additionally, the SIV nef gene is necessary for viraemia and AIDS-like pathogenesis in rhesus macaques. We report here in an in vivo murine transgenic model that thymocyte and T-cell-specific nef gene expression results in a marked decrease in thymic cellularity from 16 days post coitus. This reduction in thymocyte cell number is independent of CD4 expression and Nef-induced CD4 down-regulation, but can be restored by expressing a constitutively active p56lckF505 gene. Functional analyses have revealed a severe decrease in thymocyte and T-cell proliferation in response to both T-cell-receptor- and mitogen-mediated stimuli. In addition, a significant proportion of Nef-expressing peripheral T-cells display cell-surface characteristics associated with cellular activation. These results suggest that Nef expression in developing thymocytes can severely reduce the regeneration capacity of the immune system, whereas expression in mature T-cells dramatically decreases their potential to respond to antigen. With the recent recognition of a persistently high viral load in HIV-infected individuals, these findings have important implications for the mechanism of the progressive deterioration of the immune system that leads to AIDS.