Analysis of parameters that affect human hematopoietic cell outputs in mutant c-kit-immunodeficient mice.

Xenograft models are transforming our understanding of the output capabilities of primitive human hematopoietic cells in vivo. However, many variables that affect posttransplantation reconstitution dynamics remain poorly understood. Here, we show that an equivalent level of human chimerism can be regenerated from human CD34(+) cord blood cells transplanted intravenously either with or without additional radiation-inactivated cells into 2- to 6-month-old NOD-Rag1(-/-)-IL2Rγc(-/-) (NRG) mice given a more radioprotective conditioning regimen than is possible in conventionally used, repair-deficient NOD-Prkdc(scid/scid)-IL2Rγc(-/-) (NSG) hosts. Comparison of sublethally irradiated and non-irradiated NRG mice and W(41)/W(41) derivatives showed superior chimerism in the W(41)-deficient recipients, with some differential effects on different lineage outputs. Consistently superior outputs were observed in female recipients regardless of their genotype, age, or pretransplantation conditioning, with greater differences apparent later after transplantation. These results define key parameters for optimizing the sensitivity and minimizing the intraexperimental variability of human hematopoietic xenografts generated in increasingly supportive immunodeficient host mice. Exp Hematol 2017 Apr; 48:41-49

Identification and characterization of primitive fetal hematopoietic cells

Identification of phenotypes of human hematopoietic cells that display long-term mature cell outputs in vitro and repopulating capability in immunodeficient mice has been important to anticipating the therapeutic potential of fresh harvests of bone marrow or cord blood before or after their physical or genetic manipulation. Characterizing additional properties of these cells and elucidating the mechanisms that regulate their ability to sustain mature blood cell production has thus remained a major focus of interest. Previous studies have shown that fetal and adult human cells with long-term blood cell output potential are highly enriched in their respective GPI80+ and CD49f+ subsets of a developmentally preserved CD45+CD34+CD38-CD45RA-CD90+ population. The so-called “GPI80” hematopoietic cells found in first trimester human fetal liver are of particular interest because of their very high regenerative capability compared to their adult or even neonatal (cord blood) “CD49f” counterparts. However, in vitro conditions that enable the extensive innate self-renewal capability of either of these cell types to be maintained have remained elusive. The goals of this project were first to test the hypothesis that retention of the high regenerative activity of the GPI80+ cells in vitro would be optimized under different conditions than those that regulate their viability or mitogenesis; and second, that the GPI80+ cells of interest would co-express CD49f. The results show first, that the defining long-term cell output function of human hematopoietic stem cells (HSCs) in fetal liver is best maintained in vitro by FLT3L alone despite its poor ability to support their concomitant survival and proliferation. Secondly, co-expression of CD49f within the GPI80+ population identifies a subset with reduced short-term myeloid colony-forming activity in semi-solid medium, and greater progeny outputs in both 12-week growth factor-supplemented stromal co-cultures, and in transplanted immunodeficient mice. These findings demonstrate CD49f is a pervasive marker of human HSCs throughout ontogeny and aging. They also reinforce an increasing body of evidence that the molecular maintenance of potent regenerative potential, even in the first HSCs to appear during human development, is adversely affected by exclusive exposure to factors that most strongly stimulate their mitogenesis and even their survival.Medicine, Faculty ofGraduat

Dependence of human cell survival and proliferation on the CASP3 prodomain

Abstract Mechanisms that regulate cell survival and proliferation are important for both the development and homeostasis of normal tissue, and as well as for the emergence and expansion of malignant cell populations. Caspase-3 (CASP3) has long been recognized for its proteolytic role in orchestrating cell death-initiated pathways and related processes; however, whether CASP3 has other functions in mammalian cells that do not depend on its known catalytic activity have remained unknown. To investigate this possibility, we examined the biological and molecular consequences of reducing CASP3 levels in normal and transformed human cells using lentiviral-mediated short hairpin-based knockdown experiments in combination with approaches designed to test the potential rescue capability of different components of the CASP3 protein. The results showed that a ≥50% reduction in CASP3 levels rapidly and consistently arrested cell cycle progression and survival in all cell types tested. Mass spectrometry-based proteomic analyses and more specific flow cytometric measurements strongly implicated CASP3 as playing an essential role in regulating intracellular protein aggregate clearance. Intriguingly, the rescue experiments utilizing different forms of the CASP3 protein showed its prosurvival function and effective removal of protein aggregates did not require its well-known catalytic capability, and pinpointed the N-terminal prodomain of CASP3 as the exclusive component needed in a diversity of human cell types. These findings identify a new mechanism that regulates human cell survival and proliferation and thus expands the complexity of how these processes can be controlled. The graphical abstract illustrates the critical role of CASP3 for sustained proliferation and survival of human cells through the clearance of protein aggregates