Stroma-conditioned medium and sufficient prestimulation improve fibronectin fragment-mediated retroviral gene transfer into human primitive mobilized peripheral blood stem cells through effects on their recovery and transduction efficiency.

Mobilized peripheral blood stem cells (PBSC) are an attractive vehicle for cancer gene therapy. However these stem cells may have a reduced proliferative capacity due to previous cytotoxic chemotherapy treatment of the patient. In addition, primitive hematopoietic stem cells (HSC) from mobilized peripheral blood are almost exclusively quiescent, which makes it hard to induce proliferation in vitro and thus to improve stable transduction of introduced genes into a sufficiently large number of primitive stem cells. In this study CD34-selected mobilized PBSC from lymphoma and myeloma patients were used as target cells for retroviral-mediated gene transfer using a clinically relevant cell- and serum-free supernatant transduction protocol. We have investigated various parameters that may contribute to an improvement of the poor transduction efficiency of the primitive HSC, including prestimulation time, the use of the carboxy-terminal fibronectin fragment CH-296, as well as stromal cell line conditioned media. Retroviral supernatant transduction in combination with CH-296 increased significantly the gene transfer efficiency as compared to supernatant alone and made the use of polycations redundant. Gene transfer of primitive HSC (cobblestone area forming cell (CAFC) week 6) was specifically improved when this procedure was preceded by a 5-day pre-culture period as compared to a 2-day transduction procedure. However, irrespective of the numerical recovery, the CAFC week 6 after retroviral transduction produced less long-term culture colony-forming cells, suggesting a loss of individual stem cell quality. The addition of stroma-conditioned media during the pre-culture period did not affect the individual CAFC quality or transduction efficiency, but increased greatly the recovery of the total number of transduced and untransduced HSC leading to larger grafts containing higher numbers of transduced stem cells.

Stroma-contact prevents loss of hematopoietic stem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells.

Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality.

A novel thrombopoietin signaling defect in polycythemia vera platelets.

The pathogenesis of polycythemia vera (PV), a disease involving a multipotent hematopoietic progenitor cell, is unknown. Thrombopoietin (TPO) is a newly characterized hematopoietic growth factor which regulates the production of multipotent hematopoietic progenitor cells as well as platelets. To evaluate the possibility that an abnormality in TPO-mediated signal transduction might be involved in the pathogenesis of PV, we examined TPO-induced protein tyrosine phosphorylation using platelets as a surrogate model system. Platelets were isolated from the blood of patients with PV as well as from patients with other chronic myeloproliferative disorders and control subjects. Impaired TPO-mediated platelet protein tyrosine phosphorylation was a consistent observation in patients with PV as well as those with idiopathic myelofibrosis (IMF), in contrast to patients with essential thrombocytosis, chronic myelogenous leukemia, secondary erythrocytosis, iron deficiency anemia, hemochromatosis, or normal volunteers. Thrombin-mediated platelet protein tyrosine phosphorylation was intact in PV platelets as was expression of the appropriate tyrosine kinases and their cognate substrates. However, expression of the platelet TPO receptor, Mpl, as determined by immunoblotting, chemical crosslinking or flow cytometry was markedly reduced or absent in 34 of 34 PV patients and also in 13 of 14 IMF patients. Impaired TPO-induced protein tyrosine phosphorylation in PV and IMF platelets was uniformly associated with markedly reduced or absent expression of Mpl. We conclude that reduced expression of Mpl is a phenotypic characteristic of platelets from patients with PV and IMF. The abnormality appears to distinguish PV from other forms of erythrocytosis and may be involved in the platelet function defect associated with PV.