Pathways that govern hematopoietic stem cell fate

Hematopoietic stem cells (HSCs) compose a rare population of undifferentiated cells, residing in the bone marrow of adult individuals, ensuring life-long maintenance and replenishment of the blood system. This fantastic achievement is possible owing to two special characteristics of the HSCs: their ability to make copies of themselves (self-renew), and their capacity to differentiate to all lineages of the blood system. The process of blood formation, hematopoiesis, is a dynamic and complicated process reliant on the strict balance between a large number of regulatory factors. Hematopoietic stem cell transplantation (HSCT) is currently used to treat hematological disorders such as leukemia. Cord blood is an easily accessible source of stem cells, however the number of HSCs extracted from one cord are not enough to successfully transplant adult patients. This limitation could be circumvented if we were capable of expanding stem cells outside the body. However, to reach this goal it is crucial to first understand how these cells are regulated in their natural environment. More knowledge is required to understand the interplay between different intrinsic and extrinsic factors participating in governing HSCs. Ex vivo HSC expansion would not only be beneficial for making HSCT accessible to a larger number of patients, but would also enable profound studies of HSC function and regulation. In this thesis we have identified and evaluated factors involved in the regulation of HSC fate decisions. Transforming growth factor-β (TGFβ) is one of the most potent inhibitors of hematopoietic stem and progenitor cell (HSPC) proliferation in vitro. However, the complete mechanism behind the growth inhibitory effect and the precise function of this signaling pathway in vivo, is still to be unraveled. Our results in Article I suggest that Smad4 is a limiting factor for TGFβ-mediated Smad signaling critical for long-term HSC function and demonstrate that the level of Smad4 can modulate the response to TGFβ in human cells. Furthermore, we describe a negative regulatory role of the Smad signaling pathway on human HSPCs during regeneration after transplantation - affecting self-renewal capacity but not lineage choice. In Article II, we identify a transcriptional network, consisting of important stem cell regulators, TGFβ(Smad4)/GATA2/p57, that is critical in controlling the proliferation of primitive hematopoietic cells. We further generate a database of genes that become deregulated following TGFβ stimulation, and demonstrate that GATA2 is involved in a large part of the TGFβ response. At last, in Article III, we have studied the role of Pigment epithelium-derived factor (PEDF) in murine hematopoiesis. Our findings demonstrate that PEDF is an important regulatory factor for HSC regeneration and that PEDF in vivo works in a cell-autonomous fashion. For the first time, we propose a role of PEDF in HSC biology. Taken together, the work in this thesis has contributed to the field by increased understanding of mechanisms and factors involved in the regulation of HSC fate decisions

A network including TGFβ/Smad4, Gata2 and p57 regulates proliferation of mouse hematopoietic progenitor cells.

Transforming growth factor-β (TGFβ) is a potent inhibitor of hematopoietic stem and progenitor cell proliferation. However, the precise mechanism for this effect is unknown. Here, we have identified the transcription factor Gata2, previously described as an important regulator of hematopoietic stem cell (HSC) function, as an early and direct target gene for TGFβ-induced Smad signaling in hematopoietic progenitor cells. We also report that Gata2 is involved in mediating a significant part of the TGFβ response in primitive hematopoietic cells. Interestingly, the cell cycle regulator and TGFβ signaling effector molecule p57 was found to be upregulated as a secondary response to TGFβ. We observed Gata2 binding upstream of the p57 genomic locus, and importantly loss of Gata2 abolished TGFβ-stimulated induction of p57 as well as the resulting growth arrest of hematopoietic progenitors. Our results connect key molecules involved in HSC self-renewal and reveal a functionally relevant network regulating proliferation of primitive hematopoietic cells

Signaling via Smad2 and Smad3 is dispensable for adult murine hematopoietic stem cell function in vivo

Transforming growth factor-β (TGFβ) is a member of a large family of polypeptide growth factors. TGFβ signals mainly through the intracellular proteins Smad2 and Smad3, which are highly similar in amino acid sequence identity. A number of studies have shown that these proteins, dependent on context, have distinct roles in the TGFβ signaling pathway. TGFβ is one of the most potent inhibitors of hematopoietic stem and progenitor cell proliferation in vitro, but its role in hematopoiesis in vivo is still being determined. To circumvent possible redundancies at the receptor level and to address specifically the role of the Smad circuitry downstream of TGFβ and activin in hematopoiesis, we studied the effect of genetically deleting both Smad2 and Smad3 in adult murine hematopoietic cells. Indeed, TGFβ signaling is impaired in vitro in primitive bone marrow (BM) cells of Smad2 and Smad3 single knockout models. However, blood parameters appear normal under steady state and in the transplantation setting. Interestingly, upon deletion of both Smad2 and Smad3 in vivo, mice quickly develop a lethal inflammatory disease, suggesting that activin/TGFβ signaling is crucial for immune cell homeostasis in the adult context. Furthermore, concurrent deletion of Smad2 and Smad3 in BM cells in immune-deficient nude mice did not result in any significant alterations of the hematopoietic system. Our findings suggest that Smad2 and Smad3 function to mediate crucial aspects of the immunoregulatory properties of TGFβ, but are dispensable for any effect that TGFβ has on primitive hematopoietic cells in vivo

Bone marrow transplantation without myeloablative conditioning in a mouse model for Diamond-Blackfan anemia corrects the disease phenotype

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success

The stem cell regulator PEDF is dispensable for maintenance and function of hematopoietic stem cells

Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.Funding Agencies|Hemato-Linne grant (Swedish Research Council Linnaeus); STEMTHERAPY (Swedish Research Council); Swedish Cancer Society (Cancerfonden); Swedish Childhood Cancer Foundation; Swedish Research Council; Royal Swedish Academy of Sciences - Tobias Foundation; Lund University Hospital (ALF grant); Royal Physiographic Society in Lund (Kungliga Fysiograftska sallskapet); center of excellence grant in Life Sciences from the Swedish Foundation for Strategic Research</p