Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture.

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1-2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies

Studying global gene expression changes during hematopoietic development to identify novel regulators of hematopoietic stem cell function

Self-renewal and differentiation are the two primary attributes of all kinds of stem cells. Hematopoietic stem cells (HSCs) are multi-potent stem cells that are responsible for producing all blood cells throughout the lifetime of an organism. Hematopoietic stem cell transplantation (HSCT) is an effective method for treatment of hematopoietic disorders including leukemia and other hematopoietic malignancies, as well as immunodeficiencies. The first HSCT was performed more than 50 years ago1. Today, more than 30,000 autologous and 15,000 allogeneic transplantations are performed annually2. Bone marrow (BM), mobilized peripheral blood (MPB) and umbilical cord blood (UCB) are three major sources for HSC. Although HSC transplantation can cure a significant proportion of patients, it still has certain limitations like availability of a matched donor, graft-versus-host diseases (GvHD) and delayed engraftment post-transplantation. The process of finding donors that have compatible histocompatibility antigens (HLA) is often a challenge. In this regard, UCB derived HSCs are considered an alternative source for HSCT, it requires less stringent HLA matching as compared with BM grafts3. One of the problems related to the use of UCB as cell source is the limited number of HSCs per graft, such that one UCB unit is insufficient to treat adult patients4-6. Some approaches could be useful to overcome the limitation such as ex vivo expansion of HSCs or improving the homing capacity of HSCs. Even though a lot of information is available on various regulators that control their function in vivo, maintenance and expansion of HSCs is still a major challenge and also the biggest hurdle in order to exploit their immense potential. During development, HSCs appear in various transient sites before they finally migrate to the bone marrow, which is a major site of hematopoiesis. In adult BM, long-term repopulating HSCs are mostly quiescent and are in G0 stage of cell cycle, while an expansion of HSC pools occurs in the fetal liver during development. Therefore, we aim at studying HSCs and their niche during hematopoietic development. Both intrinsic and extrinsic factors can influence the self-renewal capacity of HSCs. In order to uncover important regulators that govern self-renewal and proliferation of HSCs, we performed genome-wide transcriptome analysis of HSCs and their niche from various stages of development using RNA-sequencing (RNA-Seq). The cell-intrinsic regulators of HSCs were shortlisted after comparing gene expression profile of HSCs isolated from fetal liver (FL) and adult bone marrow (BM) reflecting the proliferating and quiescence state, respectively. These intrinsic regulators were further validated for their functional role in hematopoiesis using a flk1:GFP/gata1:dsRed double transgenic Zebrafish line, in which the cells from blood and endothelial lineages are fluorescently labeled. Our study identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. Most of the studies on the hematopoietic niche have been done using adult BM as a model system where HSCs are mostly in a quiescent state. However, the niche in the FL has been less studied than the BM niche. Here, we performed transcriptome analysis of niche cells that directly surround HSCs by using laser capture microdissection from FL of E12.5, E14.5, and E16.5. The differentially regulated genes between FL 12.5 and FL16.5 relative E14.5 were shortlisted. From these lists of potential extrinsic factors, we identified a role of VEGF-C and S100A8 in increasing HSC number by an addition of recombinant proteins to the ex vivo culture of HSCs. Although the ex vivo expansion of HSC in the presence of these factors was achieved, These HSCs had reduced in long-term repopulation potential in vivo. The discrepancy between these findings will need further experimentation, including evaluation of homing or of possible exhaustion of HSCs in response to these factors. Lastly, we investigated the role of differentially regulated biological pathways between FL 14.5 and BM HSCs based on gene expression data obtained from RNA-seq. Interestingly, we observed that FL HSCs showed increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the tricarboxylic acid cycle (TCA). On other hand, OxPhos pathways have been demonstrated to cause exhaustion of BM HSC; but this has not been extensively studied in fetal liver HSC. We here demonstrated that FL HSCs use the OxPhos pathway in addition to glycolysis, without apparent HSC exhaustion. The results presented in this thesis form a foundation for further functional validation of novel transcriptional regulators, extrinsic growth factors and metabolic regulators in mammalian models of hematopoiesis, to ultimately provide novel and clinically relevant methods that will allow ex vivo expansion of HSCs.status: publishe

Outside-in integrin signaling regulates hematopoietic stem cells function

Integrins play an important role in haematopoietic stem cell (HSC) maintenance in the bone marrow niche. Here, we demonstrate that Periostin (Postn) via interaction with Integrin-αv (Itgav) regulates HSC proliferation. Systemic deletion of Postn results in peripheral blood (PB) anaemia, myelomonocytosis and lymphopenia, while the number of phenotypic HSCs increases in the bone marrow. Postn-/- mice recover faster from radiation injury with concomitant loss of primitive HSCs. HSCs from Postn-/- mice show accumulation of DNA damage generally associated with aged HSCs. Itgav deletion in the haematopoietic system leads to a similar PB phenotype and HSC-intrinsic repopulation defects. Unaffected by Postn, Vav-Itgav-/- HSCs proliferate faster in vitro, illustrating the importance of Postn-Itgav interaction. Finally, the Postn-Itgav interaction inhibits the FAK/PI3K/AKT pathway in HSCs, leading to increase in p27Kip1 expression resulting in improved maintenance of quiescent HSCs. Together, we demonstrate a role for Itgav-mediated outside-in signalling in regulation of HSC proliferation and stemness.status: publishe

Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

Integrins play an important role in haematopoietic stem cell (HSC) maintenance in the bone marrow niche. Here, we demonstrate that Periostin (Postn) via interaction with Integrin-alpha v (Itgav) regulates HSC proliferation. Systemic deletion of Postn results in peripheral blood (PB) anaemia, myelomonocytosis and lymphopenia, while the number of phenotypic HSCs increases in the bone marrow. Postn(-/-) mice recover faster from radiation injury with concomitant loss of primitive HSCs. HSCs from Postn(-/-) mice show accumulation of DNA damage generally associated with aged HSCs. Itgav deletion in the haematopoietic system leads to a similar PB phenotype and HSC-intrinsic repopulation defects. Unaffected by Postn, Vav-Itgav(-/-) HSCs proliferate faster in vitro, illustrating the importance of Postn-Itgav interaction. Finally, the Postn-Itgav interaction inhibits the FAK/PI3K/AKT pathway in HSCs, leading to increase in p27Kip1 expression resulting in improved maintenance of quiescent HSCs. Together, we demonstrate a role for Itgav-mediated outside-in signalling in regulation of HSC proliferation and stemness

Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1-2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.status: publishe

Distinct molecular signature of murine fetal liver and adult hematopoietic stem cells identifies novel regulators of hemapoietic stem cell function

During ontogeny, fetal liver (FL) acts as a major site for hematopoietic stem cell (HSC) maturation and expansion, whereas HSCs in the adult bone marrow (ABM) are largely quiescent. HSCs in the FL possess faster repopulation capacity as compared with ABM HSCs. However, the molecular mechanism regulating the greater self-renewal potential of FL HSCs has not yet extensively been assessed. Recently, we published RNA sequencing-based gene expression analysis on FL HSCs from 14.5-day mouse embryo (E14.5) in comparison to the ABM HSCs. We reanalyzed these data to identify key transcriptional regulators that play important roles in the expansion of HSCs during development. The comparison of FL E14.5 with ABM HSCs identified more than 1,400 differentially expressed genes. More than 200 genes were shortlisted based on the gene ontology (GO) annotation term "transcription." By morpholino-based knockdown studies in zebrafish, we assessed the function of 18 of these regulators, previously not associated with HSC proliferation. Our studies identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. In conclusion, we demonstrate that identification of genes involved in transcriptional regulation differentially expressed between expanding FL HSCs and quiescent ABM HSCs, uncovers novel regulators of HSC function.status: publishe

FACS analyses of the CD235a⁺/CD71⁺ cells from UCB, PBMNC and BM, after 5 days of expansion and 14 days of differentiation.

<p>The Q2 gate represents the population positive for CD235a (Per-CP-Cy5-5) and CD71 (PE).</p

Cytopsin of the <i>P. vivax</i> culture 24 h post-invasion for different sources of reticulocytes.

<p>A) UCB/HSPC, B) BM/HSPC, C) PB/HSPC, D) reticulocytes-enriched UCB and E) reticulocytes-enriched PB. The left panels represent pictures with a 63× magnification and the corresponding right panels represent a 100× of the left picture square. <i>P. vivax</i> infected cells are under arrow.</p