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Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38- cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.

Although the homing of hematopoietic stem cells (HSC) to the bone marrow (BM) is a crucial step in hematopoietic development and BM repopulation, the mechanisms underlying these processes have not been fully clarified. Recent studies suggest that interaction between the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor 1 (SDF-1), plays a critical role in these processes. In addition, dextran sulfate increases plasma SDF-1 levels in mice and nonhuman primates. Thus, we examined the effects of preconditioning with SDF-1 and dextran sulfate on the homing efficiency of HSCs following BM transplantation in mice. We found that the preconditioning of donor mice with either SDF-1 or dextran sulfate enhanced the homing efficiency of infused HSCs in vivo. The greatest effects were obtained with dextran sulfate. Moreover, reverse transcriptase polymerase chain reaction analysis demonstrated that SDF-1 and dextran sulfate increased transcription of a variety of homing-related genes, including those for CXCR4, lymphocyte function associated antigen-1, matrix metalloproteinase-9, very late antigen-4/5, and macrophage inflammatory protein-1. We suggest that whereas SDF-1 directly acts to upregulate CXCR4 expression in HSCs, dextran sulfate acts via multiple pathways involved in the induction of various homing-related molecules, in addition to SDF-1. Thus, preconditioning donors with dextran sulfate offers a novel clinical approach for improving the homing and engraftment of HSCs in the BM.

Gene therapy targeting hematopoietic stem cells has been proposed as a potential therapy for numerous genetic disorders affecting hematopoiesis. Moloney murine leukemia retroviral vectors are now widely used for clinical gene transfer into hematopoietic progenitors and progeny. However, maintaining expression of therapeutic genes inserted via moloney murine leukemia virus (MoMLV)-based vectors has proven to be more difficult than previously expected. In this study, an MND-IL-2R vector containing IL-2Rc gamma cDNA to treat X-linked severe combined immunodeficiency (X-SCID) was constructed from an MND vector that was modified by substituting the myeloproliferative sarcoma virus (MPSV) enhancer for that of MoMLV, deleting the negative control region located in the long terminal repeat (LTR) as an enhancer, and replacing the primer binding site (PBS) of MoMLV with the PBS of the endogenous murine retrovirus dl587rev. This vector was transduced into human CD34 + progenitor cells with comparable efficiency to that of the MoMLV-based vector. The use of this newly created vector may be advantageous for gene therapy of X-SCID.

Metachromatic leukodystrophy (MLD) is an autosomal recessive, inherited, lysosomal storage disease caused by a deficiency in arylsulfatase A (ASA). This disease is characterized by progressive demyelination leading to severe neurological symptoms. Allogenic bone marrow transplantation at an early stage of clinical course is only effective treatment currently available. Accordingly the corrective transfer of the ASA gene into hematopoietic stem cells is thought to be an important option for curative treatment for MLD. We have recently developed a selectable vector system based on ex vivo sorting of transduced cells (Migita et al. 1995). In this study, we applied this selectable system for development of MLD gene therapy. A bicistronic retroviral vector containing ASA cDNA and CD24 cDNA as a selectable marker gene was constructed. This vector was successfully transduced on fibroblasts from MLD patients, ASA activity was increased 7-fold compared to normal untransduced cells. PCR Southern analysis of hematopoietic colonies showed that transduction efficiency of CD34+ cells was 11-22%. However, after fluorescence-activated cell sorting using anti-CD24 antibody, 75-100% of colonies became vector positive. The sorting raised the ASA activity several fold compared to untransduced CD34+ progenitors. These results suggest that a bicistronic ASA vector containing a CD24 selectable marker could be a useful component of gene therapy for MLD.

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral-producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

Aims/hypothesis. Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas. Methods. Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation. Results. In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas. Conclusions/interpretation. Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed. [ABSTRACT FROM AUTHOR]