技术资料

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS),which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study,we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia,prolonged bleeding time,and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit,but not to gpIbα or gpIbβ. Therefore,we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94. View Publication

HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus,the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here,we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by textgreater10 000-fold for both CD34(+)/CD38(-) and CD34(+)/CD38(+) cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also,we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore,a TAT-SALL4B fusion rapidly expanded CD34(+) cells,and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs. View Publication

BACKGROUND Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential,a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation,the medium for 3D microcarriers was directly switched to EB medium. RESULTS hESCs on 3D microcarriers maintained pluripotency and formed EBs,which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating,EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore,this 3D system can also be adapted to human induced pluripotent stem cells,which generate functional hemangioblasts with high efficiency. CONCLUSION This 3D serum- and stromal-free microcarrier system is important for future clinical applications,with the potential of developing to a GMP-compatible scalable system. View Publication