Hematopoietic Stem Cells

Hematopoietic Stem Cells Michael E. Trigg, MD ABSTRACT. The hematopoietic system of the young child acquires, through time, the ability to cope with exposure to a number of environmental toxins and infec- tious agents. Occasionally, severe aplastic anemia occurs secondary to exposure to some of these toxins or infec- tious agents. The occurrence of severe aplastic anemia provides an opportunity to study the maturation of the hematopoietic system because often the immune system is partially intact. Hematopoietic stem cell transplants permit the study of the complete reconstitution of the hematopoietic and immunologic system. Stem cell trans- plants are often used to treat severe aplastic anemia or, alternatively, may be part of the treatment for an under- lying malignant disease or a genetic disease. Sources of stem cells and the age of the recipient and donor have an impact on the success of the stem cell transplant. A stem cell transplantation provides a window of opportunity to study and observe the normal maturation of the immune system and the sensitivity. Very clearly, children recover from severe aplastic anemia and stem cell transplanta- tions more readily with fewer problems and complica- tions than adults. The environmental risks that a child who received a stem cell transplantation faces are related primarily to the deficiencies of the hematopoietic system and immune system during the recovery phase. There- fore, diminished resistance to infectious agents, primar- ily viruses and other opportunistic organisms, are the primary risk that children who are recovering from these transplantations face. There are few data on the suscep- tibility of these children to the toxic effects of other environmental toxicants during the recovery period, which may take years before complete recovery. Pediat- rics 2004;113:1051–1057; aplastic anemia, stem cell trans- plantation, pediatrics.

steward@waksman.rutgers.edu) Accepted 3 November 2009 SUMMARY The Drosophila lymph gland, the source of adult hemocytes, is established by mid-embryogenesis. During larval stages, a pool of pluripotent hemocyte precursors differentiate into hemocytes that are released into circulation upon metamorphosis or in response to immune challenge. This process is controlled by the posterior signaling center (PSC), which is reminiscent of the vertebrate hematopoietic stem cell niche. Using lineage analysis, we identified bona fide hematopoietic stem cells (HSCs) in the lymph glands of embryos and young larvae, which give rise to a hematopoietic lineage. These lymph glands also contain pluripotent precursor cells that undergo a limited number of mitotic divisions and differentiate. We further find that the conserved factor Zfrp8/PDCD2 is essential for the maintenance of the HSCs, but dispensable for their daughter cells, the pluripotent precursors. Zfrp8/PDCD2 is likely to have similar functions in hematopoietic stem cell maintenance in vertebrates.

Retroviral induced malignancies serve as ideal models to help us better understand the molecular mechanisms associated with the initiation and progression of leukemogenesis. Numerous retroviruses including AEV, FLV, M- MuLV and HTLV-1 have the ability to infect hematopoietic stem and progenitor cells, resulting in the deregulation of normal hematopoiesis and the development of leukemia/lymphoma. Research over the last few decades has elucidated similarities between retroviral-induced leukemogenesis, initiated by deregulation of innate hematopoie- tic stem cell traits, and the cancer stem cell hypothesis. Ongoing research in some of these models may provide a better understanding of the processes of normal hematopoiesis and cancer stem cells. Research on retroviral induced leukemias and lymphomas may identify the molecular events which trigger the initial cellular transforma- tion and subsequent maintenance of hematologic malignancies, including the generation of cancer stem cells. This review focuses on the role of retroviral infection in hematopoietic stem cells and the initiation, maintenance and progression of hematological malignancies.

Introduction Hematopoietic stem cells (HSCs) sustain lifelong production of mature blood cells. In fact, it has been documented that HSCs can even outlive their original donor upon repeated serial transplantation in lethally irradiated recipients 1 , the most widely used model to study the process of HSC exhaustion. However, multiple studies have shown that serial transplantation is limited, suggesting stem cell exhaustion 1-10 . It has been documented that serial transplantation results in a permanent loss of self-renewal capacity, which is cell dose dependent 10-12 . Also, functional decline increases with repeated serial transfers 2,3,6,8,13,14 . In a competitive repopulation assay, serially transplanted stem cells showed impaired self-renewal, already after the first transplantation 8,15 . HSCs showed limited self-renewal when either purified HSCs 15 or unfractionated bone marrow cells were used 8,9 . Most of these studies have suggested that the limit to serial transplantation is caused by exhaustion of stem cells, but this has been challenged by more recent experiments in which it was argued that the engraftment defect results from an increasingly smaller number of stem cells transplanted (i.e., in vivo dilution of stem cells) 10 . In addition, it has been proposed that artifacts of the serial transplantation procedure, such as residual injury caused by removal of the HSCs from their natural environment 8,9 , might cause the decline in self-renewal capacity. This hypothesis was challenged by experiments in which animals, except for one hind limb, were repeatedly irradiated. Due to repeated stress caused by repopulation of irradiated marrow spaces, self-renewal capacity of the shielded bone marrow remained depressed and did not recover with time 16 . However, it is possible that, in conjunction with repeated proliferative stress in these shielding studies, forced cell migration and potential

Edited by Ian Wilmut, University of Edinburgh, Edinburgh, Scotland, and approved October 27, 2005 (received for review July 20, 2005) Despite two decades of studies documenting the in vitro blood- forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engi- neered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm spec- ification, increases hematopoietic progenitor formation, and, to- gether with HoxB4, enhances multilineage hematopoietic engraft- ment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice.

The use of highly purified hematopoietic stem cells as grafts is rare. 56 –58 However, the latter have the advantage of containing no detectable contaminating tumor cells in the case of autologous grafts, therefore not inducing GVHD, or presumably GVL, 139–141 in allo- geneic grafts. While they do so less efficiently than lymphocyte-containing cell mixtures, HSCs alone can engraft across full allogeneic barriers (i.e., when trans- planted from a donor who is a complete mismatch for both major and minor transplantation antigens). 139–141 The use of donor lymphocyte infusions (DLI) in the context of HSC transplantation allows for the controlled addition of lymphocytes, if necessary, to obtain or maintain high levels of donor cells and/or to induce a potentially curative GVL-response. 142,143 The main problems associated with clinical use of highly purified HSCs are the additional labor and costs 144 involved in obtaining highly purified cells in sufficient quantities.

Hematopoietic disorders are in theory curable with hematopoietic stem cell transplantation (HSCT) [1-3]. However, bone marrow (BM) and peripheral blood (PB)-HSC transplantation applications for HSCT have several draw backs, including the limitation of suitable HLA-matched donors, complications of graft versus host diseases (GvHD) and tumorigenic potential of autologous bone marrow transplant (BMT). For these reasons alternative sources of hematopoietic stem cells (HSC) such as umbilical cord blood (CB) have been explored. UCB is easily accessible and has lower GvHD incidence regardless of HLA disparity. Laghlin et al. showed that UCB HLA mismatched transplants require longer time to engraft when compared to HLA-matched sibling or unrelated marrow transplants [4]. Since UCB allows more HLA disparity than BM or PBSC grafts, finding UCB donors for minority populations who are currently under-represented in donor registries is considerably easier[5- 7].This makes UCB an attractive option when BM or PB-HSC donors are not the be best possible alternative [1]. Although the use of UCB as a stem cell source has increased significantly during recent years, especially for transplantation into children and early adulthood patients, it is not without downsides. One of the most important disadvantages of UCB as an HSC therapy is the limited available number of HSCs [8, 9]. This limitation of UCB raised considerable interest in the development of the ex vivo expansion of HSCs [8, 10, 11].

Institut Curie, Paris, France ABSTRACT While the aortic region, the para-aortic splanchnopleura/aorta-gonads-mesonephros (P-Sp/AGM) is currently considered as the source of definitive hematopoietic stem cells during development, the mouse placenta has been found to generate large numbers of these cells and to remain functional in this respect for a longer period than the P-Sp/AGM. The fetal component, which derives from the fused allantois and chorion, is responsible for this activity. We and others have shown that the pre-fusion allantois (before the stage of 6 pairs of somites) is able to yield clonogenic progenitors, provided that it is pre-cultured in toto before it is dissociated into single cells and seeded in semi-solid medium. Thus placental hematopoiesis can be concluded to derive from intrinsic precursors. It is similar in this regard to the yolk sac which both produces hematopoietic progenitors and supports their multiplication and differentiation. Hematopoietic activity, detected by in vitro colony assays, has also been recently uncovered in the human placenta. According to the data available, this newly identified source probably provides a large number of HSC during development and must play a foremost role in founding the definitive hematopoietic system.

Hematopoietic stem cells (HSCs) serve as a life-long reservoir for all blood cell types and are clinically useful for a variety of HSC transplantation-based therapies. Understanding the role of chromatin organization and regulation in HSC homeostasis may provide important insights into HSC development. Bromodomain- and PHD finger–containing protein 1 (BRPF1) is a multivalent chromatin regulator that possesses 4 nucleosome-binding domains and activates 3 lysine acetyltransferases (KAT6A, KAT6B, and KAT7), suggesting that this protein has the potential to stimulate crosstalk between different chromatin modifications. Here, we investigated the function of BRPF1 in hematopoiesis by selectively deleting its gene in murine blood cells. Brpf1-deficient pups experienced early lethality due to acute bone marrow failure and aplastic anemia. The mutant bone marrow and fetal liver exhibited severe deficiency in HSCs and hematopoietic progenitors, along with elevated reactive oxygen species, senescence, and apoptosis. BRPF1 deficiency also reduced the expression of multipotency genes, including Slamf1, Mecom, Hoxa9, Hlf, Gfi1, Egr, and Gata3. Furthermore, BRPF1 was required for acetylation of histone H3 at lysine 23, a highly abundant but not well-characterized epigenetic mark.

ABSTRACT Tissue homeostasis requires the presence of multipotent adult stem cells that are capable of efficient self-renewal and differentiation; some of these have been shown to exist in a dormant, or quiescent, cell cycle state. Such quiescence has been proposed as a fundamental property of hematopoietic stem cells (HSCs) in the adult bone marrow, acting to protect HSCs from functional exhaustion and cellular insults to enable lifelong hematopoietic cell production. Recent studies have demonstrated that HSC quiescence is regulated by a complex network of cell-intrinsic and -extrinsic factors. In addition, detailed single-cell analyses and novel imaging techniques have identified functional heterogeneity within quiescent HSC populations and have begun to delineate the topological organization of quiescent HSCs. Here, we review the current methods available to measure quiescence in HSCs and discuss the roles of HSC quiescence and the various mechanisms by which HSC quiescence is maintained.

Hematopoietic stem cells (HSCs) usage has numerous potential benefits for basic and clinical research. Growing interest in this field is caused by possible HSCs application to cure various hematologic diseases, malignancies, immunodeficiency diseases, and inborn errors of metabolism. Although the demand of HSCs is increasing, ex vivo culture of HSCs has various medium compositions. This article focuses on HSCs’ culture medium optimation in different medium supplements, which might overcome non-optimal HSCs culture results. Literature searching was conducted in PubMed and google scholar database. Paper selection processes were done by author separately and studies which met the eligible criteria were included in this review. A total of 53 relevant articles were identified and 11 articles that met the eligible criteria were included in this review. There are two main types of supplement, cytokines and non-cytokines. In cytokines addition, 7 studies generally show that the supplement support HSCs expansion. The addition of non-cytokines supplements has more diverse result in studies. Positive results with various effectivity in CD34+ expansion were shown in 3 studies, while one study shows a negative result. Medium supplementation must be explored more to find best substances which can optimize HSCs culture. Further research using non-cytokines substance shall be conducted regarding its various effect and might give us a big opportunity to find an optimal condition for HSCs culture.

* Correspondence: Lederer@cing.ac.cy; Tel.: +357-22-392764 Abstract: Accessibility of hematopoietic stem cells (HSCs) for the manipulation and repopulation of the blood and immune systems has placed them at the forefront of cell and gene therapy develop- ment. Recent advances in genome-editing tools, in particular for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) and CRISPR/Cas-derived ed- iting systems, have transformed the gene therapy landscape. Their versatility and the ability to edit genomic sequences and facilitate gene disruption, correction or insertion, have broadened the spec- trum of potential gene therapy targets and accelerated the development of potential curative thera- pies for many rare diseases treatable by transplantation or modification of HSCs. Ongoing develop- ments seek to address efficiency and precision of HSC modification, tolerability of treatment and the distribution and affordability of corresponding therapies. Here, we give an overview of recent progress in the field of HSC genome editing as treatment for inherited disorders and summarize the most significant findings from corresponding preclinical and clinical studies. With emphasis on HSC-based therapies, we also discuss technical hurdles that need to be overcome en route to clinical translation of genome editing and indicate advances that may facilitate routine application beyond the most common disorders.

R302,303A mutation (KI/KI) that prevents activation by PI3K have a similar angiogenic phenotype, although rare animals survive to adulthood. Although ARAP3 was first discovered in porcine leukocytes, it remains largely unstudied in hematopoiesis and hematopoietic stem cells (HSCs). In this thesis, we aim to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in hematopoiesis and HSCs using several conditional knockout (CKO) transgenic mouse models in addition to the KI/KI mutant mouse model. Here, we report that HSCs from surviving adult KI/KI bone marrow (BM) are compromised in their ability to self-renew and reconstitute recipient mice. To decipher the possible mechanisms of the KI/KI mutation, we utilize our genetic CKO models to conditionally delete Arap3 in hematopoietic cells and in several cell types within the HSC niche. Excision of Arap3 in hematopoietic cells using Vav-Cre does not alter the ability of ARAP3-deficient HSCs to provide multi-lineage reconstitution and to undergo self-renewal, suggesting ARAP3 does not play a cell-autonomous role in HSCs. Deletion of Arap3 in osteoblasts and mesenchymal stromal cells using Prx1-Cre resulted in no discernable phenotypes in hematopoietic development or HSC homeostasis in adult mice. However, reverse transplantation into Arap3;Prx1 CKO mice resulted in an expanded phenotypic HSC compartment, suggesting ARAP3 plays a crucial role in the BM niche to maintain and regulate HSC self-renewal. In contrast, deletion of Arap3 using VEC-Cre resulted in embryonic lethality, yet HSCs from surviving adult mice were largely normal and reverse transplantation into Arap3;VEC CKO mice revealed HSC frequencies and functions comparable to control mice. Taken together, this thesis work suggests that despite a critical role for ARAP3 in embryonic vascular development, its loss in endothelial cells minimally impacts HSCs in adult BM; however ARAP3 may play a pivotal role in the mesenchymal and osteoblastic BM niche to regulate HSC functions.

number of strategies are being investigated to overcome the cell-dose barrier, including ex-vivo expansion of UCB stem cells, intraosseous infusion of UCB cells, activating homing receptors (such as CXCR4), and use of double cord blood units [9-11]. To date, attempts at ex vivo ex- pansion of hematopoietic stem cells have been met with only limited success. To overcome the cell–dose barrier, the combination of two UCB units is becoming common- place in adolescent and adult populations [11]. The use of multiple UCB has yielded many more UCB transplants to treat adults, and this should be considered an important advance in the field of UCB transplantation [9,10]. How- ever, when two or more UCBs are transplanted into one recipient, it is common for only one to be engrafted into the patient [9-11]. The engrafting UCB unit cannot be predetermined and molecular mechanisms surrounding this finding are still unclear. In some studies, the use of two UCB units appears to have a positive impact on out- comes; however, delayed engraftment continues to pose a significant risk for patients and increased GVHD is also noted [9-11] . An additional disadvantage of using mul- tiple units is substantial increases in costs compared to the use of one UCB unit[11]. A possible way to improve outcome and extend applicability of UCB transplantation is via ex vivo or in vivo expansion and enhancement of homing.

Michelle Nguyen-McCarty Peter Klein Hematopoietic stem cells (HSCs) are able to self-renew and to differentiate into all blood cells. HSCs reside in a low-perfusion niche and depend on local signals to survive and to maintain the capacity for self-renewal. HSCs removed from the niche can survive if they receive hematopoietic cytokines, but they then lose the ability to self-renew. However, we showed previously that simultaneous inhibition of glycogen synthase kinase-3 (GSK-3) and mammalian target of rapamycin complex 1 (mTORC1) maintains HSC function ex vivo without the need for exogenous cytokines. As these experiments were initially done in heterogeneous cell populations, I then showed that purified HSCs can also be maintained under these conditions, demonstrating a direct effect of GSK-3 and mTORC1 inhibition on HSCs. Although Wnt/β-catenin signaling downstream of GSK-3 is required for this response, the downstream effectors of this network remained otherwise undefined. I therefore explored targets downstream of GSK-3 and mTORC1. I found that HSCs express a pro-autophagic gene signature and accumulate LC3 puncta only when both mTORC1 and GSK-3 are inhibited, identifying autophagy as a signature for a signaling network that maintains HSCs ex vivo. In contrast, I did not find evidence to support a role for other downstream targets of mTORC1, such as protein translation and mitochondrial biogenesis. I also report a significant reduction in total RNA content in cultured HSCs and describe a method to perform transcriptional profiling of these cells.

(Received 21 June 2016; accepted 27 June 2016) Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated im- mune remodeling. Among others, the DNA damage theory of aging of HSCs is well estab- lished, based on the detection of a significantly larger amount of gH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the he- matopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of gH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright Ó 2016 ISEH - International Society for Experimental Hematology. Pub- lished by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http:// creativecommons.org/licenses/by-nc-nd/4.0/ ).

Umbilical cord blood (UCB) has been used for transplantation in regenerative medicine of hematological disorders. The use of hematopoietic stem cells for regenerative medicine have been largely unsuccessful due to the inability to create sufficient stem cell numbers and also to excessive differentiation of them (1). In vitro studies showed that control of HSCs self-renewal in culture is difficult. Hematopoietic cytokines have failed to support reliable amplification of HSCs in culture; it seems additional factors to be required. Recently factors such as feeder layer have been reported to effect to HSC expansion. Mesenchymal stem cells as a feeder layer have been able to prevent apoptosis of expanded hematopoietic stem cells derived from cord blood (2).

Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indis- pensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strik- ingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumu- lated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16 Ink4a or p19 Arf deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.

Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Kras ex3op ) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture.

Hematopoietic stem cells (HSCs) are responsible for the daily production of all blood and immune cells in the body and have been widely used in transplantation to treat patients with leukemia, lymphoma, some solid cancers, and autoimmune diseases [4]. Although freshly isolated HSCs are known to have a very slim possibility of escape from immune rejection upon allogeneic trans- plantation, current evidence suggests that in vivo the HSC niche provides an immune-privileged site for HSCs.