Expression and activation of caspase-3/CPP32 in CD34+ cord blood cells is linked to apoptosis after growth factor withdrawal

Saini

et al. 2002

Blood

104

Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/ severe combined immunodeficient (NOD/ SCID) repopulating cell (SRC). Coculture of human ABM CD34 ؉ cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34 ؉ cells, induced more than 95% of the CD34 ؉ CD38 ؊ subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34 ؉ cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34 ؉ cells to be 1 in 9.9 ؋ 10 5 cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 ؋ 10 5 cells. All mice that received transplants of HUBECcultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow. IntroductionThe development of ex vivo culture methods that promote the expansion of adult human bone marrow (ABM) stem cells would have direct application in clinical gene therapy and stem cell transplantation. However, results obtained from stroma-based 1 and stroma-free ex vivo culture systems [2][3][4][5] have been disappointing, owing to insufficient activation of primitive CD34 ϩ CD38 Ϫ cells, cell differentiation, and a loss of repopulating capacity following short-term culture. 6 Moreover, increased CD34 ϩ cell numbers, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) are not quantitative indicators of in vivo repopulating potential. [7][8][9][10] Therefore, the importance of evaluating ex vivo cultured cells in an in vivo repopulation model has been emphasized. 8 The nonobese diabetic/severe combined immunodeficient (NOD/ SCID) model system has been used to measure the long-term reconstitution potential of ex vivo-expanded human lymphohematopoietic stem cells. 7-10 SCID-repopulating cells (SRCs) are enriched in human cord blood (CB) as compared with adult ABM and mobilized peripheral blood 11,12 and are most highly concentrated within the CD34 ϩ CD38 Ϫ population. 8 SRCs are considered to be biologically more primitive than assayable LTC-IC and CFC progenitors, 1,9,10,13 which are found in both the CD34 ϩ CD38 ϩ and the CD34 ϩ...

Section: Discussionmentioning

confidence: 99%

Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow

Saini

et al. 2002

Blood

104

“…Expanded cells were capable of faster engraftment at the expense of long-term hematopoietic reconstitution. Additional preclinical studies suggest that ex vivo expansion interferes with engraftment by introducing defects that promote apoptosis, [32][33][34] disrupt marrow homing, [35][36][37] and initiate cell cycling 38,39 (Table 2).…”

Section: Assessment Of Engraftmentmentioning

confidence: 99%

Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche

Hofmeister

Zhang

Knight

et al. 2006

Bone Marrow Transplant

203

172

Umbilical cord blood transplantation (UCBT) in adults is limited by the small number of primitive hematopoietic stem cells (HSC) in each graft, resulting in delayed engraftment post transplant, and both short-and longterm infectious complications. Initial efforts to expand UCB progenitors ex vivo have resulted in expansion of mature rather than immature HSC, confounded by the inability to accurately and reliably measure long-term reconstituting cells. Ex vivo expansion of UCB HSC has failed to improve engraftment because of resulting defects that promote apoptosis, disrupt marrow homing and initiate cell cycling. Here we discuss the future of ex vivo expansion, which we suggest will include the isolation of immature hematopoietic progenitors on the basis of function rather than surface phenotype and will employ both cytokines and stroma to maintain and expand the stem cell niche. We suggest that ex vivo expansion could be enhanced by manipulating newly discovered signaling pathways (Notch, Wnt, bone morphogenetic protein 4 and Tie2/angiopoietin-1) and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) in a manner that promotes HSC expansion with less differentiation. Improved methods for ex vivo expansion will make UCBT available to more patients, decrease engraftment times and allow more rapid immune reconstitution post transplant.

“…Since caspase-3 is activated when UCB CD34 ϩ cells are deprived of growth factors 35 and caspase-3 activation is inhibited by survivin in cancer cells, 24 the relationship between survivin expression and caspase-3 activation in growth factor-starved UCB CD34 ϩ cells was examined. Cells were cultured in the absence of growth factors in the presence of 10% HI-FBS for 48 hours, and survivin and active caspase-3 were examined by multivariate flow cytometry ( Figure 3B).…”

Section: Survivin Expression and Apoptosis In Ucb Cd34 ؉ Cellsmentioning

confidence: 99%

Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34+ cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis

Fukuda

Pelus

2001

Blood

202

215

The inhibitor-of-apoptosis protein survivin is expressed in most cancers and leukemias and during fetal development, but not in most normal adult tissues. Survivin expression was analyzed in umbilical cord blood (UCB) and adult bone marrow CD34 ؉ cells and in the factordependent MO7e cell line; also investigated was whether survivin expression was regulated by hematopoietic growth factors. Survivin messsenger RNA (mRNA) and protein were expressed in fresh UCB and marrow CD34 ؉ cells. The combination of thrombopoietin, Flt3 ligand, and stem cell factor upregulated survivin expression in CD34 ؉ cells within 24 hours; survivin expression was cell-cycle related and highest during G 2 /M, whereas growth-factor withdrawal resulted in de IntroductionApoptosis is an essential process for cell and tissue homeostasis, and apoptotic effector molecules and disordered apoptosis are involved in various diseases and neoplasias. Survivin is a newly described member of the inhibitor-of-apoptosis (IAP) family characterized by one or more highly conserved baculovirus IAP repeat (BIR) domains. [1][2][3][4] In contrast to other members of the IAP family, which are widely expressed in human tissues, 5,6 survivin is expressed primarily in fetal but not adult tissues, and its expression is aberrantly enhanced in most cancers, including carcinomas of the lung, colon, pancreas, prostate, breast, and stomach, and in most hematopoietic malignancies. 2 Furthermore, survivin is the only IAP whose expression is cell-cycle dependent. 7 Survivin suppresses apoptosis triggered by chemotherapeutic agents, tumor necrosis factor-␣/Fas ligand-induced stimuli, or caspases 3, 7, and 9, 8,9 and elevated expression correlates with poor prognosis in patients with solid tumors, acute leukemia, and lymphoma. 3,4,[10][11][12][13][14][15][16] Overexpression of survivin confers partial factor independence in interleukin-3 (IL-3)-dependent Baf/3 cells 2 and promotes cellcycle progression in hepatocarcinoma cells. 17 Targeting of survivin by antisense or dominant-negative strategies in several transformed cell models results in induction of apoptosis. [18][19][20][21][22] The enhancement of cell survival by survivin results from its binding to the mitotic spindle during G 2 /M in transformed cells, 23 probably in a complex with Cdk4 and p21, 24 which blocks caspase-3 activation. 23,25,26 These findings have led to the belief that survivin overexpression may provide a survival or proliferation advantage for cancer cells and that, because of survivin's limited expression, disrupting survivin interactions may represent a novel cancer therapy. 27 The pattern of survivin expression during development 1 suggests that it may be a general regulator of mitosis, preserving cell fidelity during cell division. However, little is known about regulation of survivin expression in normal adult cells, particularly by growth factors. Survivin is not expressed in most adult human tissues, 2,18 but expression of the mouse survivin homolog, TIAP, is related to proliferation in t...