Blood paper -----new

samhusam

Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.
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samhusam

Manipulation of hematopoietic stem/progenitor cells (HSPCs) ex vivo is of clinical importance for stem cell expansion and gene therapy applications. However, most cultured HSPCs are actively cycling, and show a homing and engraftment defect compared with the predominantly quiescent noncultured HSPCs. We previously showed that HSPCs make contact with osteoblasts in vitro via a polarized membrane domain enriched in adhesion molecules such as tetraspanins. Here we show that increased cell cycling during ex vivo culture of HSPCs resulted in disruption of this membrane domain, as evidenced by disruption of polarity of the tetraspanin CD82. Chemical disruption or antibody-mediated blocking of CD82 on noncultured HSPCs resulted in decreased stromal cell adhesion, homing, and engraftment in nonobese diabetic/severe combined immunodeficiency IL-2γ(null) (NSG) mice compared with HSPCs with an intact domain. Most leukemic blasts were actively cycling and correspondingly displayed a loss of domain polarity and decreased homing in NSG mice compared with normal HSPCs. We conclude that quiescent cells, unlike actively cycling cells, display a polarized membrane domain enriched in tetraspanins that mediates homing and engraftment, providing a mechanistic explanation for the homing/engraftment defect of cycling cells and a potential new therapeutic target to enhance engraftment.+ |% g- n" E: i

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samhusam

Hematopoietic stem cell engineering at a crossroads .) {  ~5 x3 `- V$ R% q7 Q8 h
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The genetic engineering of hematopoietic stem cells is the basis for potentially treating a large array of hereditary and acquired diseases, and stands as the paradigm for stem cell engineering in general. Recent clinical reports support the formidable promise of this approach but also highlight the limitations of the technologies used to date, which have on occasion resulted in clonal expansion, myelodysplasia, or leukemogenesis. New research directions, predicated on improved vector designs, targeted gene delivery or the therapeutic use of pluripotent stem cells, herald the advent of safer and more effective hematopoietic stem cell therapies that may transform medical practice. In this review, we place these recent advances in perspective, emphasizing the solutions emerging from a wave of new technologies and highlighting the challenges that lie ahead.

samhusam

NF-Y is necessary for hematopoietic stem cell proliferation and survival .: v9 D; J4 X, @5 ^" P" w5 [
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HSC function depends on the tight control of proliferation and the balance between self-renewal and differentiation. Here, we report that the trimeric transcription factor NF-Y is critical for the survival of cycling, but not quiescent HSCs. With the use of a conditional knockout mouse model, we demonstrate that NF-Ya deletion creates an accumulation of HSCs in G2/M and prompts apoptosis, causing hematopoietic failure and death of the animal. These defects are accompanied by the dysregulation of multiple genes that influence cell cycle control (cyclin b1 and p21), apoptosis (Bcl-2), and self-renewal (HoxB4, Notch1, Bmi-1) and are independent of p53. Our results identify NF-Y as a pivotal upstream participant in a regu-latory network necessary for the pre-servation of cycling HSCs.

samhusam

Regulation of murine hematopoietic stem cell quiescence by Dmtf1 .  a* I8 Z& }8 a1 r

! o, I' \7 S" G6 E; |+ K% \The cell-cycle status of hematopoietic stem cells (HSCs) is tightly regulated, most likely to balance maintenance of stem-cell status through quiescence and expansion/differentiation of the hematopoietic system. Tumor-suppressor genes (TSGs), with their cell cycle–regulatory functions, play important roles in HSC regulation. The cyclin-D binding myb-like transcription factor 1 (Dmtf1) was recently recognized as a TSG involved in human cancers by repressing oncogenic Ras/Raf signaling. However, the role of Dmtf1 in the hematopoietic system is entirely unknown. In the present study, we demonstrate that Dmtf1 regulates HSC function under both steady-state and stress conditions. Dmtf1−/− mice showed increased blood cell counts in multiple parameters, and their progenitor cells had increased proliferation and accelerated cell-cycle progression. In addition, long-term HSCs from Dmtf1−/− mice had a higher self-renewal capacity that was clearly demonstrated in secondary recipients in serial transplantation studies. Dmtf1−/− BM cells showed hyper proliferation after 5-fluorouracil–induced myeloablation. Steady-state expression and Induction of CDKN1a (p21) and Arf were impaired in HSCs from Dmtf1−/− mice. The function of Dmtf1 was mediated by both Arf-dependent and Arf-independent pathways. Our results implicate Dmtf1 in the regulation of HSC function through novel cell cycle–regulatory mechanisms.

samhusam

The role of Skp2 in hematopoietic stem cell quiescence, pool size, and self-renewal .
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1 B  t) o) n4 h9 i# ]& a0 P. W0 R9 EAlthough the maintenance of HSC quiescence and self-renewal are critical for controlling stem cell pool and transplantation efficiency, the mechanisms by which they are regulated remain largely unknown. Understanding the factors controlling these processes may have important therapeutic potential for BM failure and cancers. Here, we show that Skp2, a component of the Skp2 SCF complex, is an important regulator for HSC quiescence, frequency, and self-renewal capability. Skp2 deficiency displays a marked enhancement of HSC populations through promoting cell cycle entry independently of its role on apoptosis. Surprisingly, Skp2 deficiency in HSCs reduces quiescence and displays increased HSC cycling and proliferation. Importantly, loss of Skp2 not only increases HSC populations and long-term reconstitution ability but also rescues the defect in long-term reconstitution ability of HSCs on PTEN inactivation. Mechanistically, we show that Skp2 deficiency induces Cyclin D1 gene expression, which contributes to an increase in HSC cycling. Finally, we demonstrate that Skp2 deficiency enhances sensitivity of Lin− Sca-1+ c-kit+ cells and leukemia cells to chemotherapy agents. Our findings show that Skp2 is a novel regulator for HSC quiescence and self-renewal and that targeting Skp2 may have therapeutic implications for BM transplantation and leukemia stem cell treatment.

samhusam

My introduction to hematology began during my postdoctoral fellowship with Alan Bernstein at the storied Ontario Cancer Institute. This was in the mid-1980s during the early days of retrovirus-mediated gene transfer and the breathless prediction that gene therapy was imminent and would be the cure of many human diseases. Prior work from this group had already developed retroviral vectors and provided the first evidence of transduction of primary murine clonogenic progenitors; my project was to develop the means to transduce the hematopoietic stem cell. Although there was intense competition to be the first group to show hematopoietic stem cell transduction, the 2 most important lessons I learned in this period were: always look back to the past to guide the directions of the future so as not to “reinvent the wheel,” and the drive to do research should come from the intrinsic value and importance of the question being asked rather than from looking over your shoulder to stay ahead of an imagined or real competitor. The project was a close collaboration with Cristina Magli, a postdoctoral fellow in Dr Bob Phillips' laboratory, who shared a wealth of stem cell experience with me. Moreover, Bob was a leading member of the hematopoiesis and immunology research program at the Ontario Cancer Institute and had carried out a series of pioneering studies in stem cell biology, and clonal tracking of repopulating cells in particular. In response to some “new” idea or experimental design that Cristina and I would come up with, our weekly laboratory meetings were often punctuated by critical observations from Bob and Alan, including “well, in 1960-1970 Till and McCulloch, Metcalf, Dexter, we, etc, carried out such-and-such experiments.” This prevented us from entering many a blind alley and deeply instilled the principle that clonal long-term repopulation assays are the only ones that mattered. Indeed, one could argue that my whole career has been spent repeating my postdoctoral project over and over again, albeit in human cells. So the lesson for new trainees is that a judicious choice of project and mentor can have benefits that last a lifetime! Because this review already describes the pathway of discovery of our work once I established my own laboratory, I will only make 2 additional points. First, time and place are essential. As the review notes, I was fortunate to establish my laboratory at a time when key technologies were sufficiently developed to enable our work. I was also fortunate to be surrounded by a remarkable group of colleagues in Toronto who set high standards of excellence to aspire to but who were also committed to creating an unusually collaborative and collegial environment with few internal competitions. Second, trainees drive research. I have been privileged to work with a wonderful group of trainees who drove all the work mentioned in the review. It was their commitment to the projects and willingness to engage in a selfless fashion to the ferment of ideas from which research directions emerged and evolved.