Homing of hematopoietic stem cells (HSC) to their bone marrow (BM) niches is crucial to clinical stem cell transplantation. However, the molecular mechanism controlling this process remains not fully understood. In this study, we aimed to explore novel regulators of HSC homing through investigating downstream signals and effector molecules of the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis. We further characterized specific functions of targeted regulators by in vitro and in vivo migration/homing assays on human cord blood (CB) CD34+ hematopoietic stem/progenitor cells. / In summary, we have provided the first transcriptome profile of CB CD34 + cells downstream of the SDF-1/CXCR4 axis. We also reported the first evidence that HSC homing was regulated by the tetraspanin CD9. By comparing the homing-related responses of CD34+ to SDF-1 and CTCE-0214, we identified RGS13 as another potential regulator of HSC homing. It is anticipated that strategies for modulating the expressions and functions of CD9 and RGS13 might improve HSC homing to their hematopoietic niches. / To investigate the transcriptional regulation provided by the SDF-1/CXCR4 axis, we performed the first differential transcriptome profiling of human CB CD34+ cells in response to a short-term exposure of SDF-1, and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family proteins, was expressed in CD34 +CD38-/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously downregulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expressions were modulated via the CXCR4, G-protein, PKC, PLC, ERK and JAK2 signals. Pretreatment of CD34+ cells with anti-CD9 mAb ALB6 significantly inhibited SDF-1-mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells were enhanced. Infusion of CD34+ cells pretreated with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID mice. There also appeared a preferential homing/retaining of untreated CD34+CD9+ cells to these niches. Our results indicate that CD9, as a downstream member of SDF-1/CXCR4 signals might possess specific and important functions in HSC homing. / We first investigated the effects of SDF-1 and its analog, CTCE-0214 (a small cyclized peptide analog of the SDF-1 terminal regions), on homing-related properties (chemotaxis, transwell migration, adhesion and actin polymerization) of CB CD34+ cells. Our results demonstrated that both SDF-1 and CTCE-0214 induced a robust actin polymerization response and improved adhesion of CD34+ cells to fibronectin. Unlike SDF-1, CTCE-0214 did not induce a chemotactic response when added to the lower chamber of the transwell system. Addition of CTCE-0214 to the upper chamber significantly improved migration of CD34+ cells to a SDF-1 gradient, but there was no preferential enhancement in the migration of specific colony-forming unit (CFU) progenitors or the more primitive CD34+CD38 -/lo subpopulation. Pre-exposure of CD34+ cells to CTCE-0214 for 4 hours promoted cell migration, whereas SDF-1 pretreatment retarded migration. To dissect the molecular mechanisms leading to the observed functional differences mediated by SDF-1 and CTCE-0214, we investigated whether the two compounds differentially regulated the expression of several known regulators of HSC migration. Flow cytometric analysis revealed that the cell surface expression of CD26, CD44, CD49d, CD49e and CD164 was not changed by either compounds. Exposure to SDF-1, but not CTCE-0214, decreased membrane expression of CXCR4 on CD34+ cells. Addition of CTCE-0214 to the upper chamber inhibited the SDF-1-induced CXCR4 downregulation in both migrated and non-migrated cell population in the transwell setting. Notably, SDF-1 and CTCE-0214 had an opposite effect on the expression level of regulator of G-protein signaling 13 (RGS13), a negative regulator of chemokine-induced responses. Treatment of CD34+ with SDF-1 for 4 hours resulted in a significant increase in RGS13 expression, whereas CTCE-0214 induced a time-dependent decrease in RGS13 expression. Our results provide the first evidence that SDF-1 and CTCE-0214 differentially regulate migration of CD34 + cells, and we speculate that this might be attributed to their differential regulation of CXCR4 and RGS13 expression. / Leung, Kam Tong. / Adviser: Karen Kwai Har Li. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 146-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Human cord blood (CB) derived circulating endothelial colony forming cells (ECFCs) display a hierarchy of clonogenic proliferative potential and possess de novo vessel forming ability upon implantation in immunodeficient mice. Since survival of ECFC post-implantation is a critical variable that limits in vivo vasculogenesis, we tested the hypothesis that activation of Notch signaling or co-implantation of ECFC with human platelet lysate (HPL) would enhance cultured ECFC vasculogenic abilities in vitro and in vivo. Co-implantation of ECFCs with Notch ligand Delta-like 1 (DL1) expressing OP9 stromal cells (OP9-DL1) decreased apoptosis of ECFC in vitro and increased vasculogenesis of ECFC in vivo. The co-culture of ECFC with HPL diminished apoptosis of ECFC by altering the expression of pro-survival molecules (pAkt, pBad and Bcl-xL) in vitro and increased vasculogenesis of human EC-derived vessels both in vitro and in vivo. Thus, activation of the Notch pathway by OP9-DL1 stromal cells or co-implantation of ECFC with HPL enhances vasculogenesis and augments blood vessel formation by diminishing apoptosis of the implanted ECFC. The results from this study will provide critical information for the development of a cell therapy for limb and organ re-vascularization that can be applied to recovery of ischemic tissues in human subjects.

The mechanisms which regulate the growth fraction of the haemopoietic stem cell (CFU-S) and granulocyte macrophage progenitor cell (GM-CFC) have been investigated. In normal murine bone marrow (NMBM) a small proportion of the CFU-S are synthesising DNA (-10%). In contrast, in the bone marrow from mice regenerating after treatment with cytotoxic drugs and in developing haemopoietic tissues such as murine fetal liver a large proportion of the CFU-S (-40%) are synthesising DNA. Medium conditioned by normal murine and human bone marrow cells inhibited the proliferation of rapidly cycling CFU-S from regenerating bone marrow. This inhibitor was contained in a 50-100K daltons ultrafiltration fraction. In contra-distinction medium conditioned by human fetal liver cells stimulated the proliferation of CFU-S from NMBM. The stimulator was produced by adherent cells and was contained in a 30-50K daltons ultrafiltration fraction. An alternative assay for the humoral regulators of CFU-S proliferation was developed. Different numbers of haemopoietic cells were injected into lethally irradiated mice. Five days later they were injected with 2iCi of 125IUdR and sacrificed 2 hours later. There was a linear relationship between the log 125IUdR uptake into the spleen and femur and the log cell dose injected. Pre-treatment of haemopoietic cells with an S-phase specific cytotoxic drug resulted in a reduction in the 125IUdR incorporation into the spleen. This enabled the kinetic properties of a haemopoietic stem cell population to be assessed and the humoral 111 factors which modulate the growth fraction of these cells to be investigated. At early stages of gestation (11-14 weeks) in human fetal liver few GM-CFC are synthesising DNA, whereas later in gestation (>14 weeks) a large proportion of GM-CFC are in S-phase, Moore and Williams (1973b). Incubation of NMBM GM-CFC (approx 40% in DNA synthesis) with a supernatant from an early human fetal liver (11-14 weeks) reduced the proportion synthesising DNA to <5%. In contrast, the proportion of murine GM-CFC synthesising DNA was not affected by incubation with a supernatant from a late human fetal liver (>14 weeks). GM-CFC that had been switched out of cycle by incubation with a supernatant from an early gestation human fetal liver were switched back into cycle following incubation with a late human fetal liver supernatant. The inhibitor and stimulator of GM-CFC proliferation were both produced by non-adherent cells and were contained in >100K and 30-50K daltons ultrafiltration fractions repectively. It is likely that changes in the relative levels of a proliferation inhibitor and stimulator throughout gestation might control the proportion of GM-CFC in cycle.

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Molecular Mechanisms of Hematopoietic Stem Cell Development- The Role of Retinoic Acid Signaling Bhaskar Chanda For the Doctor of Philosophy Medical Biophysics University of Toronto 2013 Abstract During mouse embryonic development, the formation of blood or hematopoiesis occurs in multiple phases. The first phase or primitive hematopoiesis generates a restricted subset of blood cell lineages but is devoid of lymphoid and hematopoietic stem cell (HSC) potential. The next phase of hematopoiesis, also known as definitive hematopoiesis, is characterized by its ability to generate multilineage hematopoietic progenitors and HSCs from a specialized population of endothelial cells known as hemogenic endothelium (HE). Such endothelial to hematopoietic transitions (EHT) have been recently observed at a clonal level, however, molecular mechanisms that underlie EHT leading to the specification of HSCs have remained poorly understood. Here we show that retinoic acid (RA) signaling plays a pivotal role in embryonic hematopoiesis and HSC development. RA signaling inhibits primitive hematopoiesis, and promotes definitive hematopoiesis. This inductive effect of RA signaling extends to the specification of HSCs. Activation of the RA signaling pathway ex vivo in AGM-derived HE dramatically enhanced the repopulating potential, whereas its conditional inhibition in vivo abrogated HSC development. These repressive and inductive effects of RA signaling were mediated primarily via retinoic acid receptor (RAR)- &alpha;. We further analyzed the mechanistic basis of RA signaling with a combined use of cellular, molecular and biochemical assays, and show that &beta;-catenin dependent Wnt signaling is the downstream mediator of RA signaling. Collectively, this thesis provides new insight into molecular mechanisms that control embryonic hematopoiesis and identify the RA pathway as a key regulator of definitive hematopoiesis and HSC specification.

Amplification of hematopoietic stem cells (HSCs) from human cord blood has applications for a variety of cell therapy protocols. The purpose of this thesis (performed in collaboration with ViaCell, Inc.) was to analyze differential gene expression (especially related to cytokines) during the process of human HSC amplification in vitro. When applied to markers previously shown to be specific for HSC's and/or progenitor cells, the analysis validates ViaCell's cellular product. Total cellular RNA was isolated from cord blood samples at various stages of amplification and used to synthesize cDNAs as probes for hybridization arrays. mRNA candidates increased in cell populations enriched for stem cells were first identified using hybridization arrays, then confirmed by RT-PCR. Restriction mapping confirmed RT-PCR amplicons. The results identified several developmentally interesting cytokines (CD117, Jagged-2, Manic Fringe, and Notch) upregulated in stem cell enriched fractions. Analysis of one candidate previously shown to be a marker for HSCs and progenitors, CD117, was extended using Western blots to show a CD117-related protein upregulation. The observed upregulations did not contain many inflammatory cytokines, which could hinder survival of HSC grafts. The future hope for the non-CD117 candidates is as potential growth modifiers for stem cell samples isolated by clonogenic amplification.

Regulation of hematopoiesis through the finite control of hematopoietic stem cell (HSC) quiescence and proliferation is critical to the health of the organism since disturbances in blood production can lead to clinical malignancies such as anemia or leukemia. Therefore, elucidating the processes that govern HSCs is vital to advance our understanding of hematological diseases. Interestingly, HSCs can be regulated through a variety of ways. Extrinsic cues from the niche provide signals that govern HSC quiescence, proliferation, self-renewal, and differentiation. These external signals are converted to internal messages through the use of signal transduction pathways that relay information from the cytoplasm to the nucleus. While many pathways contribute to HSC regulation, the PI3K/AKT/mTOR-pathway is especially pertinent because it has been implicated in cell survival, metabolism, proliferation, and death. Many groups have identified key players within PI3K/AKT/mTOR-signaling that regulate HSC quiescence; however, these studies are hindered by the redundancy of multiple isoforms and compensatory signaling mechanisms by other members within the pathway. PDK1 is considered to be a master regulator of PI3K-signaling because it is directly activated by PI3Ks and can govern activity of a variety of substrates within the PI3K-pathway. Because of this, it is an excellent candidate to fully delineate how PDK1-mediated PI3K-signaling functions to maintain HSC quiescence. In the current study, conditional deletion of PDK1 in HSCs was achieved through the use of a hematopoietic specific Vav-Cre line. The loss of PDK1 in HSCs led to reduced numbers and an inability to provide radioprotection in primary BMTs. Furthermore, PDK1-deficient HSCs exhibit impaired quiescence and increased cycling. Strikingly, PDK1-mutant HSCs have markedly high levels of reactive oxygen species (ROS), which led to increased proliferation, DNA damage, and apoptosis in progenitor cells. Administration of a ROS scavenger, N-acetyl cysteine (NAC) partially rescued the increased proliferation and differentiation of phenotype Pdk1Hem-KO cells both in vitro and in vivo, suggesting that abnormal HSC activity in PDK1-deficient cells was in part due to increased ROS. Furthermore, mechanistic studies identified a remarkable decrease in the levels of nuclear HIF1α in HSCs. Immunoblots and phosflow studies demonstrated reduced activation of p-p70S6K, a well defined positive regulator, and increased GSK3β, a key negative regulator of the HIF1α protein. These data suggested that ROS levels are unrestrained since HIF1α is not present in Pdk1Hem-KO HSCs to activate transcription of genes that moderate oxidative stress. In addition, Pdk1Hem-KO HSCs also show increased levels of Hif3α and IPAS mRNA, which are believed to inhibit the transcriptional activation of HIF1α. In essence, the results from these experiments are the first to implicate PDK1 as a critical regulator of HSC quiescence and as a moderator of PI3K-signaling to alleviate oxidative stress. In addition, this study is the first to suggest that HIF3α and IPAS may play a role in HSCs.

Hematopoietic stem cells facilitate lifelong production of a diverse repertoire of functional mature blood cells. They are a critical biological reservoir that enable organisms to endure physiological challenges such as inflammation, disease, and age. The functional maintenance of hematopoietic stem cells depends not only on intrinsic cell pathways, but also on extrinsic cues that guide core behaviors like homing and self-renewal. Careful study of these extrinsic regulatory networks can deepen our appreciation of fundamental stem cell biology and motivate therapeutic approaches to treat hematologic disease. Here I show how derangement of the bone marrow regulatory environment perturbs normal hematopoiesis, and demonstrate the dependence of hematopoietic stem cells on a circulating endocrine factor.

"February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 171-209). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.