Recombinant Self-Complementary Adeno-Associated Virus Serotype Vector-Mediated Hematopoietic Stem Cell Transduction and Lineage-Restricted, Long-Term Transgene Expression in a Murine Serial Bone Marrow Transplantation Model

2008 ◽  
Vol 19 (4) ◽  
pp. 376-383 ◽  
Author(s):  
Njeri Maina ◽  
Zongchao Han ◽  
Xiaomiao Li ◽  
Zhongbo Hu ◽  
Li Zhong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Transgene Expression ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Virus Serotype ◽  
Transplantation Model

scholarly journals IGF-1-mediated osteoblastic niche expansion enhances long-term hematopoietic stem cell engraftment after murine bone marrow transplantation

Stem Cells ◽  
2013 ◽  
Vol 31 (10) ◽  
pp. 2193-2204 ◽  
Author(s):  
Anna Caselli ◽  
Timothy S. Olson ◽  
Satoru Otsuru ◽  
Xiaohua Chen ◽  
Ted J. Hofmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Hematopoietic Stem Cell ◽  
Marrow Transplantation ◽  
Hematopoietic Stem ◽  
Niche Expansion ◽  
Murine Bone Marrow ◽  
Cell Engraftment

scholarly journals Loss of hematopoietic stem cell self-renewal after bone marrow transplantation

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 872-875 ◽  
Author(s):  
P Mauch ◽  
S Hellman
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Dose Dependent Decrease ◽  
Dose Dependent

Abstract The quality of long-term hematopoietic engraftment after bone marrow transplantation (BMT) has not been well characterized. Clinical autologous BMT involves removal of less than 5% of the total content of the recipient marrow followed by ablation of the remaining marrow and reinfusion. To study long-term consequences of transplanting limited numbers of BM stem cells further, we evaluated the hematopoietic reserve in recipient animals after transplantation of varying quantities of BM. Recipient animals demonstrated a donor BM cell dose- dependent decrease in stem cell content and self-renewal capacity that was not reflected in peripheral blood (PB) counts or BM cellularity. This decrease was observed after initial BM recovery and did not change with time after transplantation, demonstrating a permanent loss in BM self-renewal capacity. In addition, animals alive at 3 months, a time selected to allow BM recovery, also demonstrated a donor BM cell dose- dependent decrease in survival at 1 year. These results emphasize the importance of optimizing stem cell number in BMT.

scholarly journals Loss of hematopoietic stem cell self-renewal after bone marrow transplantation

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 872-875 ◽  
Author(s):  
P Mauch ◽  
S Hellman
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Dose Dependent Decrease ◽  
Dose Dependent

The quality of long-term hematopoietic engraftment after bone marrow transplantation (BMT) has not been well characterized. Clinical autologous BMT involves removal of less than 5% of the total content of the recipient marrow followed by ablation of the remaining marrow and reinfusion. To study long-term consequences of transplanting limited numbers of BM stem cells further, we evaluated the hematopoietic reserve in recipient animals after transplantation of varying quantities of BM. Recipient animals demonstrated a donor BM cell dose- dependent decrease in stem cell content and self-renewal capacity that was not reflected in peripheral blood (PB) counts or BM cellularity. This decrease was observed after initial BM recovery and did not change with time after transplantation, demonstrating a permanent loss in BM self-renewal capacity. In addition, animals alive at 3 months, a time selected to allow BM recovery, also demonstrated a donor BM cell dose- dependent decrease in survival at 1 year. These results emphasize the importance of optimizing stem cell number in BMT.

DNMT3b’s Role in Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1406-1406
Author(s):  
Matthew J Boyer ◽  
Feng Xu ◽  
Hui Yu ◽  
Tao Cheng
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Transplantation ◽  
Peripheral Blood ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract DNA methylation is an epigenetic means of gene regulation and is carried out by a family of methyltransferases of which DNMT1 acts to maintain methylation marks following DNA replication and DNMT3a and DNMT3b methylate DNA de novo. DNMT3b has been shown to be essential for mammalian development and necessary for differentiation of germline and neural progenitor cells. Mutations of DNMT3b in humans lead to a rare autosomal recessive disorder characterized by immunodeficiency, centromeric instability, and facial abnormalities. We have shown by real-time, RT-PCR that DNMT3b mRNA is uniquely over-expressed by approximately 30-fold in immunophenotypically-defined longterm repopulating hematopoietic stem cells (HSCs) that are CD34−lineage−c-kit+Sca-1+ as compared to progenitor and differentiated cell types within the bone marrow and with respect to the other members of the DNMT family, namely DNMT1 and DNMT3a. To determine DNMT3b’s function in HSCs competitive bone marrow transplantation was undertaken. Isolated lineage− enriched bone marrow cells were transduced with a retroviral backbone based on the Murine Stem Cell Virus (MSCV) carrying either GFP and a short, hairpin RNA (shRNA) targeting DNMT3b or GFP alone. Following transduction 1×105 GFP+ cells along with 1×105 competitor cells were transplanted into 9.5 Gray irradiated congenic recipients. Two months following transplantation mice receiving bone marrow cells transduced with DNMT3b shRNA showed a significantly lower engraftment of donor cells as a percentage of total competitor cell engraftment in the peripheral blood as compared to those receiving cells transduced with GFP alone (24.8 vs 3.7, p<0.05) which persisted at 3 months (22.8 vs 1.5, p<0.05). Similarly, within the donor derviced cells in the peripheral blood there was a lower percentage of myeloid (CD11b+) cells at 2 and 3 months in the recipients of DNMT3b shRNA transduced cells as compared to controls. However there was no observed difference in the percentage of peripheral B (CD45R+) or T (CD3+) cells within the donor-derived cells. To determine the mechanism behind the observed engraftment defect with DNMT3b knockdown we cultured GFP+ transduced bone marrow cells in vitro with minimal cytokine support. As a control for our targeting methodology we also transduced bone marrow cells from mice harboring two floxed DNMT3b alleles with a MSCV carrying Cre recombinase and GFP. While lineage− bone marrow cells transduced with GFP alone increased 10-fold in number over two weeks of culture, cells in which DNMT3b was down regulated by shRNA or Cre-mediated recombination only doubled. Culture of lineage− bone marrow cells in methylcellulose medium by the colony-forming cell (CFC) assay revealed increases in the granulocytic and total number of colonies with DNMT3b knockdown or Cre-mediated recombination of DNMT3b similar to the increased myeloid engraftment of DNMT3b shRNA transduced cells observed 1 month following competitive bone marrow transplantation. However when 5,000 of these cells from the first CFC assay were sub-cultured there was a significant loss of colony forming ability within all lineages when DNMT3b was targeted by shRNA or Cre-mediated recombination. Taken together with the decreased engraftment of DNMT3b shRNA cells following competitive bone marrow transplantation, the observed limited proliferation in liquid culture and loss of colony forming ability during serial CFC assays is suggestive of a self-renewal defect of HSCs in the absence of DNMT3b, that was previously only reported in the absence of both DNMT3a and DNMT3b. Further elucidation of this proposed self-renewal defect is being undertaken and results of ongoing studies including long-term culture initiating cell (LTC-IC) assays and identification of genomic sites of DNA methylation within different hematopoietic subsets will also be presented.

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