Characterization of ‘Early’ vs ‘Late’ Endothelial Progenitor Cells (EPCs) Which Are Derived from Human Umbilical Cord Blood (HCB) during Ex Vivo Expansion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1706-1706
Author(s):  
Eun-Sun Yoo ◽  
Jee-Young Ahn ◽  
Yun-Kyung Bae ◽  
Seung-Eun Lee ◽  
Sang min Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Tube Formation ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord ◽  
Vascular Injuries ◽  
Rt Pcr ◽  
Different Types

Abstract EPCs have been isolated from adult peripheral blood and bone marrow. Recently, several groups reported that two types (‘early’ & ‘late’) of EPC could be isolated from peripheral blood and bone marrow when pertinent cocktails of cytokines were used. Interestingly, early and late EPCs are different in terms of expression of surface markers, the abilities of tube formation in vitro and the capabilities of re-vascularization on hind limb ischemia models in mice. We found EPC formation during ex vivo expansion of HCB and one EPC could be found from 314 CD34+ cells from HCB based on limiting dilutional assay (ref. Stem Cells; 2003, Yoo et al). However, little is known about the characteristics of ‘early’ and ‘late’ EPCs that are derived from HCB. In this study, our aims are to isolate the ‘early’ and ‘late’ EPCs from HCB during ex-vivo HCB expansion period and to characterize the biologic properties between ‘early’ and ‘late’ EPCs. 1 x 108 mononuclear cells were plated on a 100mm culture dish coated with 50ug/ml of human fibronectin (Calbiochem) and cultured in EGM-2 BulletKit system (Clonetics). Endothelial cells were assessed by colony counts, flow cytometry, proliferation assay, RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCs were also measured by in vitro transmigration assay in the presence of VEGF and SDF-1. In results, early spindle-shaped cells (‘early’ EPCs) which were grown at first week of culture were positive for CD31, CD14 and CXCR-4. Cobblestone shaped cells (‘late’ EPC) were in peak growth at second and third weeks of culture and were also positive using above antibodies except CD14. Early EPCs had not expressed mRNA of KDR, vWF and VE-Cadherin by RT-PCR. However, late EPCs expressed high level of mRNA of those endothelial marker genes. Both early and late EPCs expressed mRNA of eNOS. Late EPC produced more nitric oxide and formed more capillary tubes than those of early spindle-shaped cells. Early EPCs were readily migrated by VEGF and SDF-1 compared with those of late EPCs. In conclusions, we have found two different types of EPCs with different biologic properties during HCB ex vivo expansion. These findings may have potential clinical applications for “cell therapy” on vascular injuries (ie, hindlimb ischemia and myocardial infacrtion). Murine models for vascular injuries are being established to test the efficacy of different types of EPCs from HCB in our Lab.

scholarly journals Ex Vivo Expansion of Functional Human UCB-HSCs/HPCs by Coculture with AFT024-hkirreCells

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Muti ur Rehman Khan ◽  
Ijaz Ali ◽  
Wei Jiao ◽  
Yun Wang ◽  
Saima Masood ◽  
...  
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Critical Role ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34+cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF-βwith other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34+CD38−cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

scholarly journals Ex Vivo Expansion and Characterization of Human Umbilical Cord Blood CD34+ Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5795-5795
Author(s):  
Yu Zhang ◽  
Bin Shen ◽  
Wenhong Jiang ◽  
Zhihua Ren ◽  
Wei Dai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Scid Mice ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Surface Markers ◽  
Post Transplantation ◽  
Cd34 Cells

Abstract Background: Human umbilical cord blood (hUCB) has been considered as an alternative source of hematopoietic stem cells (HSCs) for allogeneic cell transplantation owing to its easy availability and less stringent requirement for HLA matching. However, the absolute number of HSCs in hUCB is much smaller than that in bone marrow or mobilized peripheral blood. Therefore, there is a need to develop a reliable and efficient approach for the expansion of HSCs of hUBC in vitro. Method: CD34+ cells were purified from hUCB using magnetic-activated cell sorting system and cultured in IMDM basal medium supplemented with cytokine cocktails. CD34+ cell population was identified by flow cytometry after in vitro expansion. Nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice were randomly allocated into four groups (n=4 for each group). Twenty-four hours after exposure to sub-lethal irradiation of 60Co, CD34+ cells obtained directly from hUCB or from in vitro expansion were transplanted into NOD/SCID mice. Four groups of mice were injected with saline, 0.4 million unexpanded CD34+ cells, 0.4 million in vitro expanded CD34+ cells, and 2.9 million in vitro expanded CD34+ cells, respectively. Multi-lineage differentiation was assessed using antibodies against a panel of cell surface markers (e.g., CD34, CD45, CD11, CD19, CD15, CD71, CD66, CD41a, and CD42b) at 1, 3, and 8 weeks post transplantation. Circulation peripheral blood was also collected for various analyses. Result: Human CD34+ cells were increased about 7.3 folds after 4 days’ culture in vitro. For in vivo mouse study, no cells expressing human cell surface marker were detected in mice injected with saline (negative control). One week after injection with CD34+ cells, human surface markers including CD34, CD45, and CD15 were easily detectable in all three groups. Three weeks after cell injection, CD34+ cells were decreased below 1% in group 2 and 3 but not in group 4. On the other hand, CD45+ and CD15+ populations were increased in groups 2, 3, and 4. There was a strong correlation between the numbers of injected CD34+ cells and differentiated cell populations. This trend was also observed with cells expressing megakaryocytic marker CD41 and erythroid progenitor cell marker CD71 in mouse groups 2 to 4. Eight weeks post transplantation, human CD34+ cells were hardly detected in any groups. However, peripheral blood and bone marrow cells expressing human cell surface markers of various hematopoietic lineages were detectable in groups 2-4. These results indicated that CD34+ cells from hUCB either before or after expansion in vitro are capable of differentiation into hematopoietic cells of various lineages in vivo. Our study also indicates that in vitro expanded CD34+ cells are successfully engrafted to bone marrow of NOD/SCID mice. Conclusion: An efficient experimental method has been developed for the expansion of hUBC CD34+ cells in vitro and these cells can be potentially explored for various applications in the clinic. Disclosures Jiang: Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

In Follicular Lymphoma (FL), γδt-Lymphocytes (γδT) Are Present and Expandable from Peripheral Blood and Rare in Tumour Lymph Nodes, Mostly in Peri-Follicular Areas.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1773-1773
Author(s):  
Mounia Braza ◽  
Therese Rousset ◽  
Majda Saifi ◽  
Guillaume Cartron ◽  
Sylvie Lafaye de Micheaux ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
B Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
B Cell Malignancies ◽  
Γδt Cells ◽  
Cd8 Cells ◽  
The Mean

Abstract Background: The tumour microenvironment plays an important role in the biology of FL. Different cell populations have been explored, including T-regulatory lymphocytes, macrophages, and T-cell subpopulation. The involvement of γδT in lymph nodes from FL patients or from inflammatory diseases has been rarely documented. So far, their histological pattern and prognosis significance are unknown and must be defined in order to develop new therapeutic programs including in vivo or ex vivo expansion of γδT, as developed particularly in B-cell malignancies by us and different groups. In this study, we analyzed from FL patients 1/the number of circulating γδT and their ex vivo expansion, 2/ the presence and distribution of γδT in tumour lymph nodes, and different chemokines, in comparison to inflammatory lymph nodes (ILN), by immunochemistry. Patients and Methods: Circulating γδT cells were counted in peripheral blood from patients having FL by FACS analysis. Blood samples from 34 patients were collected and expanded in vitro by using γδT ligands, referred to as “phosphoantigens”, including IPH1101 (used in clinical trials) and interleukin 2. Tumour samples from 51 patients (35 at diagnosis and 16 at relapse) having FL were collected from a single institution. Immunochemistry was used to study numbers and distribution of CD8, γδT cells, and the expression of CCL19, 21 and SDF1 chemokines. CCR7/γδT cells were analyzed by double immunofluorescence. Results were compared to 28 samples from patients having ILN. Results: The mean of circulating γδT was 0.36% (0.03–2.5) representing a mean of 2.2% of the CD3 cells. The mean percentage of γδT cells obtained after in vitro culture was 85% (2.1–95) with a mean 220-fold expansion (2-1050). The median number of γδT cells (cells/mm2) in FL lymph node was 18 versus 47.5 in the ILN (mean: 30 versus 82,5), P<.00001. The median of CD8 cells was 1235 in FL as compared to 1503 in ILN (mean: 1290 versus 1524). CD8+ cells had different localization (i.e. intra-and /or extra-follicular localization), but γδT were strictly peri-follicular in both clinical situations. Immunohistochemistry of the high endothelial venules (HEVs) and lymphatic vessels (LV) of 14 FL and 14 ILN were performed. We observed a significant difference (P= 2.10−7) in the expression of only the CCL19 chemokine between FL and ILN, with a poor staining for CCL19 in FL lymph nodes. CCL21 and CXCL12 do not present a difference in their expression levels. The stroma was reactive for all these chemokines, while the SDF1/CXCL12 chemokine shows a topographic difference in the distribution of stromal cells around HEV. Conclusions: These observations suggest that γδT cells are present and expandable in PB from patients having FL including patients with advanced disease. In addition, γδT are not abundant in lymph nodes of patients with FL compared to ILN, but γδT conserve their CCR7+ phenotype. This deficiency could be explained by migratory problems provoked by a lack of CCL19 chemokine expression. As γδT have been demonstrated to kill tumour cells, including B-malignant cells, they could be considered as essential targets for immune therapy in different cancers including B-cell malignancies, but their activation and trafficking has to be considered.

Ex Vivo Expansion of CD34+ Cells From Cord Blood, Bone Marrow, and Mobilized Peripheral Blood Using NANEX Nanofiber Scaffold

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4816-4816
Author(s):  
Stephen L Fischer ◽  
Jacqueline M Fonseca ◽  
Yukang Zhao ◽  
Linda L. Kelley ◽  
Ramasamy Sakthivel
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cell Phenotype ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Mobilized Peripheral Blood ◽  
Stem Cell Phenotype

Abstract Abstract 4816 Hematopoietic stem cell (HSC) transplantation has become the standard of care for patients with hematologic cancers, anemia, and a variety of other malignant and non-malignant disorders, with greater than 50,000 such procedures being performed globally each year, according to the Worldwide Network for Blood and Marrow Transplantation. Although mobilized peripheral blood (MPB) has become a preferred source of HSCs for transplants, bone marrow (BM) and umbilical cord blood (UCB) are also frequently utilized. Regardless of source, several groups have reported that grafts containing lower total nucleated cell (TNC) and CD34+ cell doses contribute to delayed engraftment and higher graft failure rate. Therefore, methods to increase the total cell number while maintaining the progenitor phenotype, especially the CD34+ progenitor cells, from individual grafts would have a significant clinical impact. Ex vivo expansion of HSCs prior to transplantation is one approach that offers tremendous promise for increasing cell doses and improving clinical outcomes. In many ex vivo culture systems, HSCs are cultured as a suspension cells and cultured in the presence of various media additives that act to enhance cell proliferation while reducing differentiation. An often-overlooked factor influencing fate decisions is the interaction of HSCs with a substrate. In the natural bone marrow microenvironment, HSCs maintain close contact with a complex network of stromal cells and extracellular matrix, likely indicating that cell-cell and cell-matrix interactions play an important role in maintaining their stem cell phenotype. With the goal of mimicking the bone marrow stem cell niche, Arteriocyte, Inc. has developed a 3-D NANEX nanofiber based cell culture substrate. The functionalized NANEX substrate is designed to provide topographical and substrate-immobilized biochemical cues that act in synergy with media additives to enhance HSC proliferation while maintain the progenitors stem cell phenotype. Here, we present our recent work with the NANEX platform towards comparing and achieving a high yield ex vivo expansion of CD34+ cells from MPB, BM, and UCB. Additionally, through the use of flow cytometry and CFU assays, we quantify and characterize NANEX-expanded cells from each source. Furthermore, we compared NANEX to a variety of commercially available products and demonstrate that NANEX significantly improves expansion and reduces phenotype loss during ex vivo culture. Our data indicates that NANEX technology provides a robust ex vivo expansion of HSCs and, with further GMP and clinical development, offers great potential for clinical applications. Disclosures: No relevant conflicts of interest to declare.

Peripheral Blood T Cells Generated After Allogeneic Bone Marrow Transplantation: Lower Levels of Bcl-2 Protein and Enhanced Sensitivity to Spontaneous and CD95-Mediated Apoptosis In Vitro. Abrogation of the Apoptotic Phenotype Coincides With the Recovery of Normal Naive/Primed T-Cell Profiles

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1803-1813 ◽  
Author(s):  
Nadia Chafika Hebib ◽  
Olivier Déas ◽  
Matthieu Rouleau ◽  
Antoine Durrbach ◽  
Bernard Charpentier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Bax Protein

Abstract T-cell reconstitution after bone marrow transplant (BMT) is characterized, for at least 1 year, by the expansion of populations of T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were obtained at various times after transplantation (from 45 to ≥730 days) and were tested for susceptibility to spontaneous apoptosis and anti-Fas triggered apoptosis in vitro. Substantial proportions of CD4+ and CD8+ cells generated during the first year after transplantation, but not by day 730, exhibited in these assays decreased mitochondrial membrane potential (▵Ψm) and apoptotic DNA fragmentation. The apoptotic phenotype tended to disappear late in the follow-up period, when substantial absolute numbers of naive (CD45RA+/CD62-L+) T cells had repopulated the peripheral blood compartment of the BMT patients. The rate of spontaneous cell death in vitro was significantly correlated with lower levels of ex vivo Bcl-2 protein, as assessed by cytofluorometry and Western blot analysis. In contrast, the levels of Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax ratios. Cell death primarily concerned the expanded CD8+/CD45R0+ subpopulation, although CD45R0− subpopulations were also involved, albeit to a lesser extent. These results show that the T-cell regeneration/expansion occurring after BMT is accompanied by decreased levels of Bcl-2 and susceptibility to apoptosis.

EPCs Derived from Ex Vivo Expansion of Unmobilized Human Peripheral Blood Co-Express Hematopoietic and Endothelial-Specific Markers and Can Be Derived from Culture of Highly Purified Populations of CD14-Positive Monocytes.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2682-2682
Author(s):  
Emerson E. Sharpe ◽  
Amylynn A. Teleron ◽  
Bin Li ◽  
Pampee P. Young
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
Adherent Cells ◽  
Hematopoietic Stem ◽  
Endothelial Markers ◽  
Cell Based Therapy

Abstract An increasing amount of data has suggested a more dynamic role of vasculogenesis, whereby bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) home and contribute to new blood vessel formation during tumor growth, ischemic injury, and wound healing. EPCs can be obtained by isolating hematopoietic progenitor cells from BM or cord blood. Alternatively, ex vivo expansion of unmobilized human peripheral blood (PB) can generate adherent cells, PB-EPCs, that express endothelial markers and also, upon administration, incorporate into developing neovasculature. The relative ease of obtaining unmobilized human PB has made PB-EPCs an attractive candidate with which to develop cell based therapy to treat ischemia. In parallel with clinical trials designed to understand their therapeutic potential, there is a continued effort to better characterize the PB-EPC and understand its biology. It is currently thought that EPCs are directly derived from a CD34+/lin- faction of hematopoietic stem cells (HSCs). However, in our current study, we have confirmed prior reports that ex vivo expansion of human PB generates similar numbers of EPCs as compared to plating unfractionated human BM, which contains >50-fold higher CD34+/lin- content, suggesting that PB-EPCs may not be derived from the CD34+/lin- population. We used immunofluorescence and FACs analysis to further show that PB-EPCs not only express endothelial markers such as vWF, Vascular Endothelial Growth Factor Receptor 1 and 2 (also known as flt-1 and flk-1, respectively), VE-cadherin, UEA-1 lectin, Tie-1 and Tie-2 but also hematopoietic markers such as CD45 and CD14, a marker enriched on monocytes. To test if PB-derived CD14-positive cells can give rise to PB-EPCs, we isolated them from human PB to >98% purity and plated them on fibronectin-coated coverslips. In vitro culture of CD14-positive cells generated adherent clusters of spindle shaped cells morphologically similar to EPCs. Culture of the CD14-negative fraction failed to yield any adherent cells. After ten days, the coverslips were removed and the cells were stained with various endothelial (flk-1, vWF, uptake of DiI-AcLDL, and UEA-1 lectin) and monocyte/hematopoietic (CD14 and CD45) cell markers. In analysis of these slides, the EPCs derived from the purified CD14 fraction stained positive for all six markers. These observations suggest that PB-EPCs can differentiate from cells of the monocytic lineage in vitro without the necessity of interaction from cells contained in the CD14-negative population. Further experiments will test the possibility that monocytes may be an intermediate in the differentiation of EPCs in vivo.

Direct Evidence for Ex Vivo Expansion of Human Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 798-798
Author(s):  
Kiyoshi Ando ◽  
Takashi Yahata ◽  
Tadayuki Sato ◽  
Hiroko Miyatake ◽  
Hideyuki Matsuzawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
Direct Evidence ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (Exp Hematol, 27:904–915, 1999), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Peripheral Blood T Cells Generated After Allogeneic Bone Marrow Transplantation: Lower Levels of Bcl-2 Protein and Enhanced Sensitivity to Spontaneous and CD95-Mediated Apoptosis In Vitro. Abrogation of the Apoptotic Phenotype Coincides With the Recovery of Normal Naive/Primed T-Cell Profiles

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1803-1813 ◽  
Author(s):  
Nadia Chafika Hebib ◽  
Olivier Déas ◽  
Matthieu Rouleau ◽  
Antoine Durrbach ◽  
Bernard Charpentier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Bax Protein

T-cell reconstitution after bone marrow transplant (BMT) is characterized, for at least 1 year, by the expansion of populations of T cells with a primed/memory phenotype and by reverse CD4/CD8 proportions. T lymphocytes from 26 BMT patients (mostly adults) were obtained at various times after transplantation (from 45 to ≥730 days) and were tested for susceptibility to spontaneous apoptosis and anti-Fas triggered apoptosis in vitro. Substantial proportions of CD4+ and CD8+ cells generated during the first year after transplantation, but not by day 730, exhibited in these assays decreased mitochondrial membrane potential (▵Ψm) and apoptotic DNA fragmentation. The apoptotic phenotype tended to disappear late in the follow-up period, when substantial absolute numbers of naive (CD45RA+/CD62-L+) T cells had repopulated the peripheral blood compartment of the BMT patients. The rate of spontaneous cell death in vitro was significantly correlated with lower levels of ex vivo Bcl-2 protein, as assessed by cytofluorometry and Western blot analysis. In contrast, the levels of Bax protein remained unchanged, resulting in dysregulated Bcl-2/Bax ratios. Cell death primarily concerned the expanded CD8+/CD45R0+ subpopulation, although CD45R0− subpopulations were also involved, albeit to a lesser extent. These results show that the T-cell regeneration/expansion occurring after BMT is accompanied by decreased levels of Bcl-2 and susceptibility to apoptosis.

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