Comparative Study of Hemopoietic Precursors from Fetal and Adult Bone Marrow: Utilization of Stem Cells Derived from Miscarriages

Michejda, Maria; Bellanti, Joseph A.; Mazumder, Amitabha; Verma, Udit; Wu, Ai Guo

doi:10.1159/000264350

Cited by 7 publications

(5 citation statements)

References 9 publications

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“…Several groups, including our own, have pursued the use of fetal cells for transplantation [12,[36][37][38][39]. Fetal stem cells are most likely to be used for in utero transplantation, but their use in children and adults may be feasible.…”

Section: Discussionmentioning

confidence: 99%

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

et al. 2001

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Objectives: Defining methods for the efficient transduction of fetal stem cells could lead to novel fetal therapies for blood cell disorders and other birth defects. In this study, we analyzed the effects of various parameters on the retroviral transduction of primitive hematopoietic progenitors/stem cells isolated from fetal liver. Methods: Candidate stem cells were isolated by fluorescence-activated cell sorting from midtrimester human livers based on the phenotype CD38–CD34++lineage– (lineage = glycophorin A, CD3, CD14, CD19, CD20 and CD56). A murine retroviral vector with a truncated human low-affinity nerve growth factor receptor (ΔNGFR) gene was used to transduce the candidate stem cells. Marker gene expression was monitored by flow cytometry using an anti-NGFR mAb. Candidate stem cells were transduced immediately after isolation or after up to 4 days of culture in serum-deprived medium containing the growth factors kit ligand and granulocyte-macrophage colony-stimulating factor. The effects on transduction efficiency of the addition of 4 µg/ml protamine sulfate and/or centrifugation to concentrate the candidate stem cells and virus were tested. After transduction, the cells were expanded for 10–21 days before determining the frequency of NGFR+ cells among the different hematopoietic progeny. Results: Efficient transduction of candidate stem cells, at an average rate of 46%, was achieved after 3 days of culture with a single exposure to virus. Longer than 3 days of culture or repeated exposure to viral supernatant did not significantly improve the rate of transduction. The use of centrifugation at 1,200 g for 1 h and the addition of protamine sulfate during the transduction procedure were critical to achieving a high rate of transduction. Marker gene expression was observed on the progeny of the transduced cells in conjunction with CD34 (progenitors), glycophorin A (erythrocytes), CD14 (monocytes), CD15 (granulocytes) and CD41 (megakaryocytes). Conclusions: This study demonstrates that the efficient transduction of fetal candidate stem cells can be achieved under defined culture conditions using a retroviral vector. These results encourage further examination of in utero and ex utero gene therapy as a means of treating birth defects.

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Section: Discussionmentioning

confidence: 99%

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

et al. 2001

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“…For instance, BM harvested from the long bones of fetal specimens has been used as a source of hematopoietic stem cells (HSCs) [ 8 ] and MSCs [ 9 , 10 ] for research. These cells have also been proposed as a source of donor cells for clinical transplantation [ 11 – 13 ].…”

Section: Introductionmentioning

confidence: 99%

A quantitative assessment of the content of hematopoietic stem cells in mouse and human endosteal-bone marrow: a simple and rapid method for the isolation of mouse central bone marrow

et al. 2015

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BackgroundIsolation of bone marrow cells, including hematopoietic stem cells, is a commonly used technique in both the research and clinical settings. A quantitative and qualitative assessment of cell populations isolated from mouse and human bone marrow was undertaken with a focus on the distribution of hematopoietic cells between the central bone marrow (cBM) and endosteal bone marrow (eBM).MethodsTwo approaches to cBM isolation from the hind legs were compared using the C57BL/6J and BALB/cJ strains of laboratory mice. The content of hematopoietic stem cells in eBM was compared to cBM from mice and human fetal bone marrow using flow cytometry. Enzymatic digestion was used to isolate eBM and its effects on antigen expression was evaluated using flow cytometry. Humanized immunodeficient mice were used to evaluate the engraftment of human precursors in the cBM and eBM and the effects of in vivo maturation on the fetal stem cell phenotype were determined.ResultsThe two methods of mouse cBM isolation yielded similar numbers of cells from the femur, but the faster single-cut method recovered more cells from the tibia. Isolation of eBM increased the yield of mouse and human stem cells. Enzymatic digestion used to isolate eBM did, however, have a detrimental effect on detecting the expression of the human HSC-antigens CD4, CD90 and CD93, whereas CD34, CD38, CD133 and HLA-DR were unaffected. Human fetal HSCs were capable of engrafting the eBM of immunodeficient mice and their pattern of CD13, CD33 and HLA-DR expression partially changed to an adult pattern of expression about 1 year after transplantation.ConclusionsA simple, rapid and efficient method for the isolation of cBM from the femora and tibiae of mice is detailed. Harvest of tibial cBM yielded about half as many cells as from the femora, representing 6.4 % and 13 %, respectively, of the total cBM of a mouse based on our analysis and a review of the literature. HSC populations were enriched within the eBM and the yield of HSCs from the mouse and human long bones was increased notably by harvest of eBM.Electronic supplementary materialThe online version of this article (doi:10.1186/s12878-015-0031-7) contains supplementary material, which is available to authorized users.

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“…If embryonic cells are a necessity in research, then there is at least one researcher who has been trying to obtain and work with them in a more acceptable manner. 59 The efforts of Maria Michejda, M.D., from Georgetown University, in working with bone marrow tissue from naturally miscarried second trimester fetuses should give hope for researchers who do not want to work with morally questionable materials. 60 (In the use of fetal tissue from spontaneous miscarriages, however, certain ethical criteria still need to be respected, as explained by Spagnolo.)…”

Section: Are There Alternatives To Hek 293?mentioning

confidence: 99%

The Ethics of HEK 293

Wong¹

2006

The National Catholic Bioethics Quarterly

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Comparative Study of Hemopoietic Precursors from Fetal and Adult Bone Marrow: Utilization of Stem Cells Derived from Miscarriages

Cited by 7 publications

References 9 publications

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

High-Efficiency Retroviral Transduction of Fetal Liver CD38–CD34++ Cells: Implications for in utero and ex utero Gene Therapy

A quantitative assessment of the content of hematopoietic stem cells in mouse and human endosteal-bone marrow: a simple and rapid method for the isolation of mouse central bone marrow

The Ethics of HEK 293

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