scholarly journals Ex vivo expansion marginally amplifies repopulating cells from baboon peripheral blood mobilized CD34+ cells

2002 ◽  
Vol 117 (4) ◽  
pp. 924-934 ◽  
Author(s):  
Françoise Norol ◽  
Michel Drouet ◽  
Françoise Pflumio ◽  
Marjorie Léonardi ◽  
Frédéric Mourcin ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Cd34 Cells

Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1920-1920
Author(s):  
Santosh Saraf ◽  
Hiroto Araki ◽  
Benjamin Petro ◽  
Kazumi G Yoshinaga ◽  
Simona Taioli ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Transcript Levels ◽  
Epigenetic Modifiers ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Abstract 1920 Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2'-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures: No relevant conflicts of interest to declare.

Ex Vivo Expansion Reduces Neutropenia Post Autologous Transplantation of Peripheral Blood Hematopoïetic Stem Cells in Patients with Follicular Lymphoma. A Randomized, Double Blind Trial,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4009-4009
Author(s):  
Helene Trebeden-Negre ◽  
Sylvain Choquet ◽  
Michelle Rozenzwajg ◽  
Nabih Azar ◽  
Francois Lefrère ◽  
...  
Keyword(s):  
Stem Cells ◽  
Blood Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Autologous Transplantation ◽  
Ex Vivo Expansion ◽  
Control Group ◽  
Double Blind ◽  
Cd34 Cells ◽  
The Mean

Abstract Abstract 4009 The ex vivo culture of Hematopoietic Stem Cells (HSC) with various combinations of cytokines can increase the number of mature hematopoietic cells that are theoretically capable of rapidly release neutrophils and platelet and reduce recovery duration post transplantation. In patients, the infusion of such cells has been reported, but the short-term effect was not clear. In a randomized, double blind study, we used expanded cells from 4×106/kg peripheral blood hematopoietic selected CD34+cells in comparison to a non manipulated graft containing the same number of CD34+ cells; we designed an ex vivo expansion protocol based on a cocktail of early or late acting cytokines with different culture duration in order to obtain progenitors at various stages of differentiation 1) primitive progenitors obtained from selected CD34+cells cultured for 8 days in presence of fetal liver tyrosine kinase 3 ligand (FLT3-ligand), stem cell factor (SCF), interleukin-3 (IL-3) and thrombopoietin (TPO), 50 ng/ml, each 2) committed megakaryocyte progenitors (Mks) obtained after culture for 10 days in presence of TPO et SCF (50 ng/ml, each) or 3) committed granulocytes and megakaryocyte (GMks) progenitors obtained after culture for 10 days in presence of TPO, SCF and G-CSF (100 ng/ml). Eighteen Non Hodgkin Lymphoma patients submitted to autologous transplantation after a myeloablative regimen consisting of AraC: Day (D)-6 to D-3: 200mg/m2/12h - VP16: 100mg/m2/12h: D-6 to D-3 - BCNU: 300mg/M2: D-6- Melphalan: 140mg/M2: D-2 could be evaluated. Patients in the Expansion Group received graft with 1×106/kg non manipulated cells combined with primitive progenitors issues from 2×106/kg CD34+ cells and Mks progenitors (Mks Expansion Group) or GMks progenitors (GMks Expansion Group) coming from 1×106/kg CD34+cells; unmanipulated cells were used as a source of immunocompetent cells. In the mean, patients of control group (n=10) received 1.3±0.9×108Total Nucleated Cells (TNC)/Kg, 2.7±1.2×106 CD34+/kg, 49±17×104/kg CFU-GM and 17.7±.7104CFU-Mks/kg; in the Mks (n=4) and GMks (n=4) Expansion group, they received respectively in the mean: 61.5±18.5 and 90.1±21.3×108TNC /Kg, 26.5±10.7 et 31± 11.2 x106CD34+/kg, 451± 188 et 557±216×104/kg CFU-GM, 358±212 et 39±18.1 x104/kg CFU-Mk. No cytokines were administered after transplantation. No toxicity was observed after cell infusion. The mean times to reach white blood cell (WBC) recovery (WBC >1x 109/l) was significantly shorter after administration of expanded cells, 14 (10–16), 12 (11–14) and 9 (9–10) days respectively in control, Mks and GMks Expansion Group (p=0.01). Median profound neutropenia (neutrophils<0,5×109/l) duration was 4 (4–5) days in the GMk Expansion Group versus 9 (7–11) in the Mks Expansion Group and 9 (6–14) in the control group (p<0.05); no patient demonstrated abrogation of neutropenia. In contrast, ex vivo expansion did not reduce the time to platelet recovery (>25 or50×109/L) despite numerous mature megakaryocytes and CFU-Mks in the Mks Expansion Group; that could be related to the colonies size, very small from expanded cells. No secondary hypoplasia was observed during the 12 months follow-up. This study shows that in comparison with unmanipulated cells, peripheral blood haematopoietic cells expanded from similar doses of CD34+ cells accelerate neutrophil recovery without impairing long-term haematopoiesis and open interesting perspectives in the field of allogeneic cord blood cell transplantation. Disclosures: Leblond: Roche, Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Ex vivo expansion of CD34+/CD41+ late progenitors from enriched peripheral blood CD34+ cells

2000 ◽  
Vol 79 (1) ◽  
pp. 13-19 ◽  
Author(s):  
P. Halle ◽  
C. Rouzier ◽  
J. Kanold ◽  
N. Boiret ◽  
C. Rapatel ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells

scholarly journals Effects of CD34+ cell selection and perfusion on ex vivo expansion of peripheral blood mononuclear cells

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 958-970 ◽  
Author(s):  
CE Sandstrom ◽  
JG Bender ◽  
ET Papoutsakis ◽  
WM Miller
Keyword(s):  
Cell Cultures ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Similar Distribution ◽  
Peripheral Blood Mononuclear ◽  
Cd34 Cells ◽  
Blood Mononuclear Cells

Ex vivo expansion of peripheral blood mononuclear cells (MNCs), cultured both directly and after selection for CD34+ cells, was compared in static and continuously perfused cultures containing interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G- CSF), and stem cell factor (SCF). Cultures inoculated with either MNCs or CD34+ cells produced cells that were remarkably similar after 10 days of culture, as evidence by cell morphology, expression of CD34, CD33, CD15, and CD11b, and the fractions of cells giving rise to colony- forming units granulocyte-monocyte (CFU-GM) and long-term culture- initiating cells (LTC-IC). Static and perfusion cultures gave similar average total cells and CFU-GM expansions for both MNC and CD34+ cell cultures. However, those samples that performed poorly in static culture performed at near-normal levels in perfusion. In addition, perfusion supported higher LTC-IC numbers for both MNC and CD34+ cell cultures. While total cell expansion was about ten times greater in CD34+ cell cultures (approximately 100-fold), CFU-GM expansion (approximately 20-fold) was similar for both MNC and CD34+ cell cultures. The similar distribution of cell types produced in MNC and CD34+ cell cultures allows direct comparison of total and colony- forming cell production. After 15 days in perfusion, MNC cultures produced 1.5-, 2.6-, and 2.1-fold more total cells, CFU-GM, and LTC-IC, respectively, than the same sample selected and cultured as CD34+ cells. Even if the CD34+ selection process was 100% efficient, CFU-GM production would be 1.5-fold greater for MNCs than for CD34+ cells.

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.

scholarly journals SALL4 Is a Key Factor in HDAC Inhibitor Mediated Ex Vivo Expansion of Human Peripheral Blood Mobilized Stem/Progenitor CD34+CD90+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1566-1566 ◽  
Author(s):  
Hiro Tatetsu ◽  
Fei Wang ◽  
Chong Gao ◽  
Shikiko Ueno ◽  
Xi Tian ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cd34 Cells

Abstract Hematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all types of mature cells within the blood and immune systems. Despite our progress in understanding the molecular factors that support the self-renewal and differentiation of the hematopoietic system in vivo, less is known on how to modulate the factors that govern the self-renewal of hematopoietic stem/progenitor cells (HSPCs) ex vivo. Unlike in the case of embryonic stem (ES) cells, expansion of CD34+ HSPC in culture in general is at the expense of loss of “stemness”. HSPCs can be collected from cord blood (CB), mobilized peripheral blood (PBSC), and rarely bone marrow (BM) at the present practice. Due to the limited CD34+ cell number in one single cord blood unit, much of the current efforts on developing technology of ex vivo expansion of HSPC uses cord blood as a source and is clinically applied to cord blood HSPC transplants. However, there are growing needs for expanding PBSCs for transplant-related practices such as HSPC expansion from poor autologous mobilizations, gene therapy or genome-editing via TALENs or CRISPR/Cas9. Developing a technology that would allow HSPC ex vivo expansion from both CB and PBSC sources is a key step towards this goal. Several groups have reported that ex vivo culture of CB CD34+ cells with HDAC inhibitors (HDACi) can lead to expansion of a CD34+CD90+ population, which is responsible for enhanced marrow-repopulating potential. In this study, we ask whether HDACi can have a similar effect on PBSC CD34+ cells. Furthermore, we have explored the mechanism(s) mediated by HDACi in CD34+CD90+ cell expansion. First we assessed a panel of HDACi to identify the most potent molecule for CD34+CD90+ cell expansion and selected trichostatin A (TSA) for future study. Next, TSA was added to the cytokines (SCF, Flt3 ligand, IL-3 and IL-6) to further characterize its potential in PBSC CD34+CD90+ cell expansion. We observed TSA treated CD34+ cultures with cytokines yielded 4.8 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines with DMSO solvent control. We next examined SCID repopulating ability (SRA) to evaluate the cultured CD34+CD90+ cells in vivo. We observed that mice transplanted with 3 million CD34+ cells treated with TSA had higher degree of human cell chimerism than those treated with DMSO at 8 weeks bone marrow and peripheral blood (32% vs 18%; p < 0.05), which was further confirmed by secondary transplantation. Furthermore, these cells were capable of differentiating into cells belonging to multiple hematopoietic lineages. To investigate the molecular mechanisms responsible for the expansion of functional HSCs and progenitors that were observed following TSA treatment, we analyzed the expression levels of several HSPC related genes, which were compared between CD34+ cells treated with TSA and DMSO. Significantly higher transcript levels were detected for GATA 2 (p < 0.05), HOXB4 (p < 0.05), HOXA9 (p < 0.05), and SALL4 (p < 0.05) by real time quantitative RT-PCR in TSA expanded cells as compared with controls. To evaluate whether these transcription factors can contribute to the expansion of CD34+CD90+ cells, GATA2, HOXB4 or SALL4 shRNAs were transfected into PBSC CD34+ cells, followed by culture with TSA. Among these transcription factors, knocking down SALL4 expression led to the most significant reduction of CD34+CD90+ cell numbers (33% of reduction). In addition, overexpression of SALL4 in PBSC CD34+ cells led to an increase of CD34+CD90+ cell numbers when compared to controls (p < 0.05). Overall, our study demonstrated a novel HDACi mediated ex vivo PBSC culture technology that leads to the expansion of CD34+CD90+ cells and an increase of the marrow repopulating potential of these cells. Both gain-of-function and loss-of-function studies support that SALL4 is a key transcription factor responsible for the process. Future study on the use of HDACi or other methods to increase SALL4 expression/function will be highly beneficial to ex vivo HSPC (CB and PBSC) expansion technology. Disclosures No relevant conflicts of interest to declare.

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