Topic of the Month Expansion of HSC
For any clinical application of HSCs, or other adult stem cells, expansion of the stem cells without loss of self-renewal capacity is crucial. Studied for almost 100 years, hematopoietic stem cells (HSC) are probably the best characterized adult stem cells to date. However, not much is known on how to culture human HSC, without losing their self-renewal capacity. Abundant literature testifies to in vitro (or ex vivo)
HSC expansion efforts, but progress has been sparse. In
the beginning, mostly cytokine cocktails were tested.
Heike and Nakahata give an overview of the plethora of
literature reporting on HSC expansion with the help of
cytokines. Besides an overview of the effect of the individual cytokines on HSC and progenitors, Heike and Nakahata also include a description of the different Ex vivo expansion of hematopoietic stem cells by cytokines. ABSTRACT Since these initial attempts at cytokine-driven HSC expansion, research has turned towards understanding the molecular pathways involved in HSC development, self-renewal and differentiation into hematopoietic cells. In his review, Nakano presents the latest advances in the understanding of how HSC develop, how they are regulated, how they are maintained and how these new findings are exploited to expand HSC in vitro. The review also covers approaches to derive HSC from embryonic stem cells and includes a summary table of molecules possibly involved in HSC self-renewal. Hematopoietic stem cells: generation and manipulation. ABSTRACT One emerging and promising approach to HSC expansion as well as for derivation of HSC from ES cells includes the manipulation of the Hox family of transcription factors. Particularly, HoxB4 has been previously described to expand HSC in vitro and to enhance in vivo repopulating activity of HSC, in the murine model. However, these approaches have included overexpression of the HoxB4 gene in the HSC, which may become a problem for later clinical approaches. Now, two additional papers demonstrate that delivering human HoxB4 protein in soluble form to the HSC could lead to successful in vitro expansion. In two back-to-back papers, researchers took advantage of the ability of HoxB4 protein to passively translocate through membranes and used recombinant human HoxB4 protein. Krosl et al. used a purified human TAT-HoxB4 fusion protein as an addition to their culture conditions (including IL-3, IL-6 and SCF) to expand murine HSC, and Amsellem et al. used a feeder layer of stromal cells secreting human HoxB4 to expand HSC isolated from human cord blood. Both groups describe expansion and an increase in repopulating HSCs in mouse models. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. ABSTRACT To continue on the theme of improved feeder layers, more recently, stem cell research has turned towards a better characterization of so-called stem cell niches, realizing that not all self-renewal regulating signals are intrinsic to the stem cell. This trend has also moved into HSC research and the following two papers have contributed significantly to the understanding of the, until then quite vague, HSC niche. Previous reports had suggested that the bone marrow osteoblasts, osteogenic cells lining the endosteal surfaces, may play a role in HSC regulation. Accordingly, osteoblasts have been shown to produce hematopoietic growth factors. By conditional inactivation of the BMP receptor 1A on osteoblasts, Zhang et al. were able to increase the number of osteoblasts in their mouse model and to demonstrate a concomitant increase in HSCs. The authors show a physical association of HSCs and excess spindle-shaped N-cadherin +/CD45 ─ osteoblastic cells and thus identify the HSC niche more clearly. Identification of the haematopoietic stem cell niche and control of the niche size. ABSTRACT Targeting a different pathway, Calvi et al. also demonstrate a crucial role for osteoblasts in the HSC niche. Osteoblasts are activated through parathyroid hormone (PTH) or PTH-related protein, and both proteins bind to the same receptor, PPR. By overexpressing PPR in transgenic mice, Calvi et al. obtained significantly increased numbers of HSCs in these mice. Although a significant increase in IL-6, SCF and SDF-1 could be detected in the transgenic mice, the increase in HSC numbers was dependent on membrane interactions between the osteoblasts and the HSCs. The authors were able to show an increase in Notch1 signaling in HSCs of the transgenic mice. They were further able to emulate the HSC expansion effect in vitro, by successfully expanding wild-type HSC on either stromal cells isolated from the transgenic mice, or by treating cultured wild-type stromal cells with PTH. But most importantly for future clinical applications, Calvi et al. were able to demonstrate a convincing in vivo increase of HSCs in wild-type mice, by simple daily injection of PTH. Moreover, PTH treatment of animals undergoing myeloablative bone marrow transplantation dramatically improved survival rates. Osteoblastic cells regulate the haematopoietic stem cell niche. ABSTRACT Other approaches to emulate the HSC niche are presented by the following two papers, with Li et al. hypothesizing that endothelial cells from the yolk sack and the para-aortic splanchnopleura may have the capacity to expand HSC, and Zhang et al. hypothesizing that fetal liver cells may produce factors that promote HSC expansion, as those organs are sites of active HSC expansion during development. Li et al. co-cultured murine HSCs on endothelial cells derived from the yolk sack or the para-aortic splanchnopleura and obtained a significant increase in HSC as evidenced by increases in competitive repopulating capacity of the co-cultured HSCs. Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro. ABSTRACT Zhang et al. isolated a novel fetal liver cell population that supports HSC expansion in vitro, as shown by increases in long-term repopulating capability. By analyzing gene expression in these fetal liver cells, the authors identified IGF-2 as a novel growth factor for HSCs, which can expand HSC repopulating capacity significantly when added to a standard HSC expansion cocktail. Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. ABSTRACT Danet et al. explore the hypothesis that within the bone marrow niche, HSCs are distributed along an oxygen gradient, with the primitive stem cells in the more hypoxic areas and the proliferating progenitors in the oxygen-rich areas of the bone marrow. The authors evaluated the functional and cellular responses of human adult bone-marrow derived HSCs to hypoxia in culture and demonstrate an expansion of repopulating HSC, in the murine model. Moreover, the authors observed an upregulation of angiogenic receptors and increased production of VEGF, which could be involved in in vitro HSC maintenance. Expansion of human SCID-repopulating cells under hypoxic conditions. FULL TEXT Related to HSC-intrinsic signals are emerging approaches targeting known molecular pathways. For example, it is known that cell cycle regulation is crucial to cell fate determination and thus it was shown in a murine model that the cell cycle regulator p21 cip1/waf1 is a molecular mediator of HSC quiescence. Another cell cycle inhibitor, p18 INK4C, has now been implicated in regulation of HSC fate. Yuan et al. show increased numbers of HSCs in p18 INK4C gene-deleted mice. Moreover, the authors demonstrate a strong repopulation advantage of HSC from the gene-deleted mice over HSC from wild-type mice, with no evidence of improved homing of the p18 INK4C gene-deleted HSCs. The same was observed when bone marrow cells from the p18 INK4C gene-deleted primary transplant recipients were transplanted into secondary recipients, again in competition with wild-type HSCs. In contrast to p21 cip1/waf1 gene-deleted HSCs pool which exhausts under stress conditions, p18 INK4C deficient HSC appear to self-renew. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. ABSTRACT Technical difficulties make the characterization of the HSC niche, i.e. the bone marrow environment, challenging. It is therefore possible that the complex growth requirements provided by the HSC stem cell niche may never be fully characterized, and therefore never emulated adequately in simple in vitro systems. On the other hand, approaches targeting molecular pathways within the HSC may be equally difficult to apply in the clinic. An alternative approach to expand HSC of clinical use is suggested by in vivo expansion of HSC transduced with a drug resistance gene. This approach has evolved from the efforts to derive HSC resistant to chemotherapy drugs, to prevent severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In murine models, HSC are transduced with a gene encoding O 6-alkylguanine-DNA-alkyltransferase (AGT), an enzyme which repairs alkylated DNA and confers resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This allows for in vivo enrichment of transduced cells following chemotherapy. While still far from optimized, such approaches could possibly one day allow for expansion of HSC within their natural niche and thus yield expanded HSCs that do not lose any of their capacities and are capable of life-long reconstitution of large adult humans. Using an improved lentiviral expression vector, Zielske et al. obtained a significant expansion of lentivirally tranduced human cord blood HSC in NOD/SCID mice after non-myeloablative transplant conditioning. This was achieved after only two regimens of drug selection. Taking these murine studies one step further, Neff et al. describe improved engraftment in the canine model, a well-established pre-clinical model for the testing of allogeneic transplantation strategies. Dogs were transplanted with transduced DLA (dog leukocyte antigen)-matched allogeneic CD34 + cells, after a total body irradiation preparative regimen.
To conclude, the reader avid to learn more on HSCs, should not miss the review by Lensch and Daley covering over 100 years of research on murine and human HSCs. Origins of mammalian hematopoiesis: in vivo paradigms and in vitro models. FULL TEXT for ISSCR members Updated: October 13, 2004 |
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