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To reduce the number of apheresis procedures and maintain the usual rate of hematopoietic recovery in patients treated with high-dose chemotherapy, we studied the effect of adding a small volume of ex vivo expanded bone marrow to low doses of CD34(+) blood stem cells. Thirty-four patients with breast cancer received G-CSF (10 microg/kg/day) priming followed by a limited volume (50-100 ml) bone marrow aspiration and standard 10-liter aphereses. Marrow was expanded ex vivo using the AastromReplicell system and infused along with low doses of blood-derived CD34(+) cells, collected in one apheresis. Thirty-one evaluable patients received a median CD34(+) blood stem cell dose of 0.7 x 10(6)/kg (range, 0.2-2.5) and 4.7 x 10(7) nucleated cells/kg (range, 1.98-8.7) of ex vivo expanded marrow. All patients recovered with normal blood counts and engrafted 500 neutrophils/microl and 20 000 platelets/microl in a median of 10 and 13 days, respectively. Multivariate analysis revealed that, in addition to CD34(+) lineage negative cell quantity, the quantity of stromal progenitors contained in the ex vivo expanded product correlated with engraftment outcome (r = 0.551, P = 0.004). Our results indicate that ex vivo expanded bone marrow is capable of facilitating engraftment when combined with low doses of mobilized blood derived CD34(+) cells.

It is largely unknown whether the immune repertoire can be reconstituted successfully after high-dose chemotherapy and transplantation using ex vivo expanded hematopoietic stem cell (HSC) grafts. It is critically important for the transplant outcome that immune repertoire reconstitution progresses after ex vivo expanded HSC graft transplants at least as efficiently as that seen after conventional HSC transplants. Previously, we showed that the T cell receptor V beta (TCRVB) third complementarity determining region (CDR3) diversification after ex vivo expanded bone marrow (BM) HSC graft transplants was similar to that seen after conventional peripheral blood stem cell transplants (PBSCTs). In the present study, the CDR3 diversity of the six immunoglobulin (Ig) heavy chain variable region gene (V(H)) families was examined in five breast cancer patients who were transplanted with ex vivo expanded BM HSCs as the only source of stem cells. For comparison, 12 healthy adults and four conventional PBSCT recipients were also studied. Using both CDR3 fingerprinting and single strand conformation polymorphism (SSCP) methodologies, it is shown that the contribution of the V(H) families to the overall repertoire among healthy adults is highly variable and not always proportional to V(H) family member size. After both ex vivo expanded HSC transplants and conventional PBSCTs, the V(H) CDR3 repertoires were limited in size diversity at 6 weeks post transplant. By 6 months, however, V(H) families displayed a repertoire diversity that was as complex as that seen in healthy adults. No difference was seen between ex vivo expanded HSC graft transplant recipients and conventional PBSCT recipients in V(H) repertoire diversity. In one patient there was a follow-up analysis 12 months after ex vivo expanded graft transplant, and the diversity of the V(H) families was maintained. In all patients, the amino acid size of the CDR3 regions fell within adult limits at all time points post transplant. These results indicate that B cell repertoire regeneration after ex vivo expanded hematopoietic cell graft transplants is similar to that seen after conventional PBSCT.

A 32-year-old female with WHO grade IV, dialysis dependent, lupus nephritis was treated with high-dose immunosuppression and autologous stem cell rescue. Stem cells were mobilized with cyclophosphamide (CY) and G-CSF, and 4.07 x 10(6) CD34+ cells/kg were obtained after CD34+ cell selection using the CellPro column. The preparative regimen consisted of CY, and antithymocyte globulin (ATG), with methylprednisolone. After apparent primary engraftment of neutrophils on day 9, the patient developed recurrent neutropenia on day 19. She showed no evidence of engraftment by day 35, and back-up unmanipulated stem cells were given without effect. Subsequently, she received unmanipulated peripheral stem cells (2 x 10(6) CD34+ cells/kg) from an HLA-identical sibling. The patient remained pancytopenic and expired on day 62 from disseminated fungal infection. An autopsy revealed no evidence of hematopoietic recovery. Progenitor cell assays were performed with the patient's stem cells, which were collected prior to transplantation, and serum collected day 27. Morphologic examination of the patient's cell colonies grown in the presence of her serum revealed abnormal shapes and non-adherent cells. There were significantly fewer BFU-e colonies and a trend toward fewer CFU-GM colonies with the patient's cells and serum compared to normal donor cells. We concluded that a substance present in her serum mediated graft failure and prevented engraftment after additional stem cell infusions.

In order to test if the carboxyl terminal polypeptide of the Retinoblastoma (Rb) tumor suppressor protein, could be used to suppress the growth factor-independent growth phenotype of p210bcr-abl positive myeloid cells, we introduced a truncated form of the 3' end of the Rb cDNA encoding its last 173 amino acid residues (Rb C-box) which localize into the cytoplasm where the p210bcr-abl transforming protein is found, into myeloid cells (32D) which depends on the p210bcr-abl protein for IL3 growth factor-independent growth (32D-p210). The expression of the plasmid vectors carrying the Rb C-box cDNAs was shown to inhibit the abl tyrosine specific protein kinase activity of the p210(bcr-abl) oncoprotein and to suppress the IL3-independent growth phenotype of the 32D-p210 cells. The Rb C-box polypeptides did not suppress the growth of the untransfected 32D parental cell line in methylcellulose in the presence of IL3-conditioned medium. These results suggest that the cytoplasmic localization of the p210(bcr-abl) allows it to escape the effect of intranuclear proteins such as Rb which negatively regulate the p145(c-abl) kinase.

We first showed that the introduction of a bcr-abl transcription unit into the 32D murine myeloid cell line (P210bcrabl32D) converts this cell line from an IL3 dependent cell line to an IL3 growth independent cell line. We next cloned a fragment of the bcr-abl cDNA, which codes for the bcr oligomerization domain and neighboring regions. To test for a transformation inhibitory effect of this oligomerization inhibitory peptide transcription unit on the p210bcr-abl mediated IL3 independent growth of the P210bcrabl32D cell line, we transiently co-electroporated into the growth factor dependent 32D cells, mixtures of plasmids which contained varying ratios of the plasmid expression vectors for the bcr oligomerization inhibitory peptide along with a smaller amount of the plasmid expression vector for the full length p210bcr-abl. (The P210bcr-abl protein converts the 32D from a growth factor dependent into a growth factor independent cell line.) We then showed that the oligomerization domain containing fragment from the bcr and bcr-abl proteins, can be used to inhibit the IL3 independent growth of p210bcr-abl positive 32D cells. These studies may be of eventual interest for those investigators whose goal is to design molecular therapeutic approaches to CML based on the use of peptidomimetic chemical functionalities, which mimic the structure and the inhibitory binding properties of the oligomerization domain containing fragment so as to inhibit the transforming function of the P210bcr-abl oncoprotein.