Apoptotic Regulation in Primitive Hematopoietic Precursors

Bcl-2 and bcl-xL function as suppressors of programmed cell death. The expression of bcl-2 protein in vivo is associated with long-lived hematopoietic cells such as mature lymphocytes and early myeloid progenitors. Bcl-xL, a homologue of bcl-2, is also expressed in lymphocytes and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, and bak) act by promoting apoptotic cell death as shown from their expression in hematopoietic cell lines. We analyzed the expression of bcl-2 and bcl-x proteins in hematopoietic precursors obtained from various cell sources in adult mobilized peripheral blood collected from 13 patients with solid tumors, 8 adult bone marrow, and 12 umbilical cord blood. The analysis was based on the expression of the proliferation and activation specific antigens, CD38 and class II (HLA-DR). Similarly, we analyzed the expression of bcl-2-related proteins bcl-xL, bax, bad, and bak before and during ex-vivo expansion. Hematopoietic precursors expressing strongly the CD34 antigen (CD34s+) and lacking CD38 or HLA-DR expression were analyzed by using three-color immunofluorescence staining. The majority of CD34+ cells expressed bcl-2 and unexpectedly showed a bimodal distribution of low and high expression. More cells that lacked or expressed low density CD38 expressed low bcl-2 than the more differentiated counterparts (those with high density CD38). Immaturity (ie, little or no HLA-DR) is associated with the expression of low bcl-2 compared with HLA-DR+. However, HLA-DR−/low population contained a lower number of cells expressing low bcl-2 (30% to 40%) than CD38−/low in comparable samples. The hematopoietic precursors with bcl-2low and bcl-2high formed a homogeneous population of undifferentiated lymphoid-like cells having a similar forward scatter. These cells expressed strongly the bcl-xL protein (>95%) but were bax low (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expression of apoptosis specific protein (ASP) was also low (3.4% ± 3.1%) as was Annexin V. In addition, the CD34+/CD38−showed low cell cycle activity (<2.2%). Induction of apoptosis by overnight incubation of CD34 cells in serum-deprived medium resulted in the upregulation of bcl-2 as a single population histogram. Thus, these results suggest that in quiescent hematopoietic precursors, the bcl-2 protein plays a less prominent role as a survival promoter than bcl-xL and that the low bcl-2 expression did not promote apoptosis. During day 10 of ex vivo expansion of CD34+cells in liquid culture containing stem cell factor, interleukin-3 (IL-3), IL-6, IL-1β, and erythropoietin, the CD34+/CD38− cells expressed high bcl-2 as a single population histogram, and greater than 90% were bcl-xL high. However, the expression of pro- and apoptotic antigens increased: bax (10% to 15%), bad (5% to 8%), bak (6% to 14%), and ASP (6% to 10%). These results show the importance of monitoring the expression of these proteins when defining the culture conditions for ex vivo expansion. © 1998 by The American Society of Hematology.

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Apoptotic Regulation in Primitive Hematopoietic Precursors

Blood ◽

10.1182/blood.v92.6.2041 ◽

1998 ◽

Vol 92 (6) ◽

pp. 2041-2052 ◽

Cited By ~ 64

Author(s):

Rowayda Peters ◽

Serge Leyvraz ◽

Lucien Perey

Keyword(s):

Cell Death ◽

Ex Vivo ◽

Apoptotic Cell ◽

Ex Vivo Expansion ◽

Single Population ◽

Homogeneous Population ◽

Hla Dr ◽

Cd34 Cells ◽

Related Proteins

Abstract Bcl-2 and bcl-xL function as suppressors of programmed cell death. The expression of bcl-2 protein in vivo is associated with long-lived hematopoietic cells such as mature lymphocytes and early myeloid progenitors. Bcl-xL, a homologue of bcl-2, is also expressed in lymphocytes and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, and bak) act by promoting apoptotic cell death as shown from their expression in hematopoietic cell lines. We analyzed the expression of bcl-2 and bcl-x proteins in hematopoietic precursors obtained from various cell sources in adult mobilized peripheral blood collected from 13 patients with solid tumors, 8 adult bone marrow, and 12 umbilical cord blood. The analysis was based on the expression of the proliferation and activation specific antigens, CD38 and class II (HLA-DR). Similarly, we analyzed the expression of bcl-2-related proteins bcl-xL, bax, bad, and bak before and during ex-vivo expansion. Hematopoietic precursors expressing strongly the CD34 antigen (CD34s+) and lacking CD38 or HLA-DR expression were analyzed by using three-color immunofluorescence staining. The majority of CD34+ cells expressed bcl-2 and unexpectedly showed a bimodal distribution of low and high expression. More cells that lacked or expressed low density CD38 expressed low bcl-2 than the more differentiated counterparts (those with high density CD38). Immaturity (ie, little or no HLA-DR) is associated with the expression of low bcl-2 compared with HLA-DR+. However, HLA-DR−/low population contained a lower number of cells expressing low bcl-2 (30% to 40%) than CD38−/low in comparable samples. The hematopoietic precursors with bcl-2low and bcl-2high formed a homogeneous population of undifferentiated lymphoid-like cells having a similar forward scatter. These cells expressed strongly the bcl-xL protein (>95%) but were bax low (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expression of apoptosis specific protein (ASP) was also low (3.4% ± 3.1%) as was Annexin V. In addition, the CD34+/CD38−showed low cell cycle activity (<2.2%). Induction of apoptosis by overnight incubation of CD34 cells in serum-deprived medium resulted in the upregulation of bcl-2 as a single population histogram. Thus, these results suggest that in quiescent hematopoietic precursors, the bcl-2 protein plays a less prominent role as a survival promoter than bcl-xL and that the low bcl-2 expression did not promote apoptosis. During day 10 of ex vivo expansion of CD34+cells in liquid culture containing stem cell factor, interleukin-3 (IL-3), IL-6, IL-1β, and erythropoietin, the CD34+/CD38− cells expressed high bcl-2 as a single population histogram, and greater than 90% were bcl-xL high. However, the expression of pro- and apoptotic antigens increased: bax (10% to 15%), bad (5% to 8%), bak (6% to 14%), and ASP (6% to 10%). These results show the importance of monitoring the expression of these proteins when defining the culture conditions for ex vivo expansion. © 1998 by The American Society of Hematology.

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Ex Vivo Expansion of Frozen Cord Blood Products without Selection.

Blood ◽

10.1182/blood.v104.11.2844.2844 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2844-2844

Author(s):

Ian K. McNiece ◽

Jenny Harrington ◽

Joshua Kellner ◽

Jennifer Turney ◽

Elizabeth J. Shpall

Keyword(s):

Cord Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

In Vivo Studies ◽

Adherent Cells ◽

Blood Products ◽

Cd34 Cells ◽

Duke University

Abstract Ex vivo expansion of cord blood products (CB) has been proposed as an approach to increase the number of cells available from a single CB unit. We and others have reported the requirement of CD34 selection for optimal expansion of CB products, however, the selection of frozen CB products results in significant losses of CD34+ cells with a median recovery of 43% (range 6 to 203%, N=40) and low purities resulting in decreased expansion. Therefore we explored approaches to expand CB without prior selection and have described the use of co-culture of CB mononuclear cells (MNC) on mesenchymal stem cells (MSC). In the present study we have evaluated the expansion of clinical CB products (provided by Duke University CB Bank CB). MNC were obtained after ficol separation of RBCs and 10% of the CB product was cultured on preformed layers of MSC in T150 flasks containing 50ml of defined media (Sigma Aldrich) plus 100 ng/ml each of rhSCF, rhG-CSF and rhTpo. After 6 days of culture, the non adherent cells were transferred to a Teflon bag and a further 50 ml of media and GFs added to the flask. Again at day 10, non adherent cells were transferred to the Teflon bag and media and growth factors replaced. At day 12 to 13 of incubation the cells were harvested, washed and total nucleated cell (TNC) counts and progenitor assays performed. In three separate experiments we have achieved greater than 20 fold expansion of TNC with a median of 22, and a median expansion of GM-CFC of 37 fold. Morphologic analysis demonstrated the expanded cells contained high levels of mature neutrophils and neutrophil precursors. In vivo studies in NOD/SCID mice also demonstrated that the expanded cells maintained in vivo engraftment potential. Clinical studies are being designed to evaluate the in vivo potential of CB MNC products expanded on MSC.

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

Blood ◽

10.1182/blood.v118.21.1920.1920 ◽

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.

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Activity of MK1775, a selective Wee1 inhibitor, alone or in combination with gemcitabine, in sarcomas.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10084 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10084-10084

Author(s):

Jenny Kreahling ◽

Damon R. Reed ◽

Parastou Foroutan ◽

Gary Martinez ◽

Robert Gillies ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Soft Tissue ◽

Soft Tissue Sarcoma ◽

Therapeutic Efficacy ◽

Ex Vivo ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Primary Therapy

10084 Background: Sarcomas consist of more than 50 subtypes of mesenchymal tumors. Doxorubicin alone or in combination has been the primary therapy for treatment of sarcomas; however, the response rates are suboptimal in many of the more common adult subtypes of soft tissue sarcoma. Accordingly, new agents are needed for the treatment of this heterogeneous group of diseases. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Methods: MK1775 treatment of multiple sarcoma preclinical models at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death. In our current study we have investigated the therapeutic efficacy of MK1775 in sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo in a xenograft model of osteosarcoma both alone and in combination with gemcitabine. Results: In patient-derived bone and soft tissue sarcoma samples ex vivo treatments show MK1775 in combination with gemcitabine causes significant apoptotic cell death suggesting that this treatment may represent a novel approach in the treatment of sarcomas. The cytotoxic effect of Wee1 inhibition on sarcoma cells appears to be independent of p53 mutational status. Furthermore, in a patient-derived osteosarcoma xenograft mouse model we show the therapeutic efficacy of MK1775 in vivo by utilizing magnetic resonance imaging (MRI) and diffusion MRI methods. Our data shows MK1775 in combination with gemcitabine dramatically slows tumor growth, increases apoptotic cell death and increases CDC2 activity. Cell viability, a clinically established prognostic indicator of survival, was lowest with the combination and very low in animals treated with MK1775 alone. This was mainly due to increased mineralization of the tumors. Caspase-3 was increased in MK1775 treated animals by immunohistochemistry as well. Conclusions: These results together with the promising safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent alone or in combination with gemcitabine in the treatment of both adult as well as pediatric sarcoma patients.

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Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽

10.1182/blood.v93.3.1097 ◽

1999 ◽

Vol 93 (3) ◽

pp. 1097-1105 ◽

Cited By ~ 111

Author(s):

G. Güenechea ◽

J.C. Segovia ◽

B. Albella ◽

M. Lamana ◽

M. Ramı́rez ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Progenitors ◽

Short Term ◽

Immunodeficient Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

Abstract The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

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Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells

Cellular & Molecular Biology Letters ◽

10.2478/s11658-014-0183-7 ◽

2014 ◽

Vol 19 (1) ◽

Cited By ~ 14

Author(s):

Elize Wolmarans ◽

Thandi Mqoco ◽

Andre Stander ◽

Sandra Nkandeu ◽

Katherine Sippel ◽

...

Keyword(s):

Cell Death ◽

Esophageal Carcinoma ◽

Ex Vivo ◽

Apoptotic Cell ◽

Critical Role ◽

Carcinoma Cells ◽

In Vivo Studies ◽

Apoptotic Pathway

AbstractCancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 μM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1 fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells. This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent.

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

Blood ◽

10.1182/blood.v124.21.1566.1566 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1566-1566 ◽

Cited By ~ 3

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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Incidence of Ex Vivo Expansion on the Capacity of Cord Blood Graft To Generate Immune Cells: Rational for Co-Infusion of Expanded and Non Expanded Fractions?.

Blood ◽

10.1182/blood.v104.11.407.407 ◽

2004 ◽

Vol 104 (11) ◽

pp. 407-407

Author(s):

Michelle Rosenzwajg ◽

Marie-Catherine Giarratana ◽

Jean-Claude Gluckman ◽

Luc Douay ◽

Ladan Kobari

Keyword(s):

Cord Blood ◽

Immune Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Th2 Response ◽

Gm Csf ◽

Hla Dr ◽

Cd34 Cells ◽

Significant Difference ◽

And Function

Abstract The aim of our study was to determine whether ex vivo expansion of umbilical cord blood (UCB) progenitor cells induces changes in their capacity to generate immune cells. CD34+ CB cells were cultured for 14 days with SCF, FLT3-l, TPO and G-CSF, inducing a total cells, CD34+ and LTC-ICs increase of 1500, 120 and 8 fold respectively. Non expanded (d0) and 14-day expanded (d14) CD34+ cells were compared for their capacity to produce T lymphocytes (TLs) using the fetal thymus organ culture system and DCs generated from d0 and d14 CD34+ cells were compared for their differentiation, phenotype and function. Total percentages of CD4+, CD4+CD8− and CD4+CD8+ TLs obtained from d0 and d14 CD34+ cells were comparable. In both fractions, most of the CD4+ T cells co-expressed iCD3 but a lower proportion of d14 derived TLs expressed sCD3. However, there was no significant difference between d0 and d14 derived TLs in term of Vb chain representation, all TCR-Vb chains examined being represented in each case. These data indicate that d0 and d14 CD34+ cells have a similar capacity to generate TLs and that expansion does not induced any skewing of the TCR-Vb repertoire. D0 and d14 CD34+ cells were next cultured with SCF, FL, GM-CSF and TNF-a to compare their capacity to differentiate into DCs. Similar percentages of CD1a+ DCs expressing the same levels of HLA-DR and co stimulatory molecules were obtained. DCs derived from d14 CD34+ cells were less potent to stimulate allogeneic TLs, but the pattern of cytokines produced by stimulated TLs was similar and no shift towards a predominant Th1 or Th2 response was observed. Moreover, in spite of a quantitative increase (15 fold) related to the CD34 pool amplification, we observed a decreased capacity (13-fold) of d14 cells to generate DCs compared to d0 CD34+ cells. Overall, these results indicate that ex vivo expansion of CD34+ cells doesn’t induce any major modification in T Lymphopoiesis capacity while alters somehow the capacity of the graft to generate DCs. We discuss in the context of UCB transplantation, the putative interest of co-infusion of expanded and non expanded fractions in view of improving myelopoiesis in the graft without subverting the immune reconstitution.

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Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽

10.1182/blood.v93.3.1097.403k04_1097_1105 ◽

1999 ◽

Vol 93 (3) ◽

pp. 1097-1105 ◽

Cited By ~ 4

Author(s):

G. Güenechea ◽

J.C. Segovia ◽

B. Albella ◽

M. Lamana ◽

M. Ramı́rez ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Progenitors ◽

Short Term ◽

Immunodeficient Mice ◽

Nonobese Diabetic ◽

Cd34 Cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

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Identification of functionally distinct macrophage subpopulations in Drosophila

eLife ◽

10.7554/elife.58686 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jonathon Alexis Coates ◽

Elliot Brooks ◽

Amy Louise Brittle ◽

Emma Louise Armitage ◽

Martin Peter Zeidler ◽

...

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Homogeneous Population ◽

Cell Numbers ◽

Functional Differences ◽

Heterogeneous Cell Population ◽

Wound Responses ◽

Macrophage Subpopulations ◽

And Function

Vertebrate macrophages are a highly heterogeneous cell population, but whileDrosophilablood is dominated by a macrophage-like lineage (plasmatocytes), until very recently these cells were considered to represent a homogeneous population. Here, we present our identification of enhancer elements labelling plasmatocyte subpopulations, which vary in abundance across development. These subpopulations exhibit functional differences compared to the overall population, including more potent injury responses and differential localisation and dynamics in pupae and adults. Our enhancer analysis identified candidate genes regulating plasmatocyte behaviour: pan-plasmatocyte expression of one such gene (Calnexin14D) improves wound responses, causing the overall population to resemble more closely the subpopulation marked by theCalnexin14D-associated enhancer. Finally, we show that exposure to increased levels of apoptotic cell death modulates subpopulation cell numbers. Taken together this demonstrates macrophage heterogeneity inDrosophila, identifies mechanisms involved in subpopulation specification and function and facilitates the use ofDrosophilato study macrophage heterogeneity in vivo.

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