Nicotinamide Modulates Ex-Vivo Expansion of Cord Blood Derived CD34+ Cells Cultured with Cytokines and Promotes Their Homing and Engraftment in SCID Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 725-725 ◽  
Author(s):  
Tony Peled ◽  
Sophie Adi ◽  
Iddo Peleg ◽  
Noga G. Rosenheimer ◽  
Yaron Daniely ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Scid Mice ◽  
Cell Phenotype ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Nicotinamide (NA) is a non-competitive inhibitor of NAD(+)-dependent ADP-ribosyl transferases, of CD38 NADase (a major regulator of cellular NAD levels) and of Sir2 histone-deacetylase. These enzymes are playing a pivotal role in regulation of signal transduction pathways and gene expression. In the present study, we evaluated the effect of NA on the ex-vivo expansion of cord-blood (CB) derived CD34+ cells and their bone-marrow (BM) homing and engraftment potential. Culturing of CD34+ cells for three weeks in the presence of cytokines (SCF, TPO, IL-6, FLT3-ligand) only or cytokines + NA (5mM) resulted in similar expansion of CD34+ cells (40-fold relative to input). However, a remarkable increase in the fraction of CD34+ cells displaying an early progenitor cell phenotype (CD34+Lin−) was observed in the NA-treated cultures as compared with cytokines-only treated cultures (18.6 ± 3% and 0.7 ± 0.06%, n=6, p<0.05, respectively). Tracking the cell-cycle history by PKH2 staining showed fewer division cycles of CD34+ cells cultured with NA. These results may suggest a direct correlation between the rate of proliferation and expansion of CD34+Lin− cells. NA-treated CD34+ cells express similar levels of CXCR4 but display increased migratory activity in response to CXCL12 over CD34+ cells treated with cytokines only (36 ± 19% and 11 ± 4%, n=4, p<0.05, respectively). In order to test their homing potential, similar number of mononuclear cells (MNC), before or following expansion with or without NA, were labeled with CFSE and transplanted into irradiated NOD/SCID mice. Twenty-four hours later the numbers of human cells (CD45+CFSE+) and human progenitor cells (CD34+CFSE+) in the BM were counted. Homing of CD45+CFSE+ cells was comparable in the three groups tested. However, CD34+CFSE+ cells with BM homing potential were 3-fold more numerous in NA-treated cultures relative to cytokines-treated cultures, and 6-fold more than in non-cultured CB cells (n=14, p<0.05). To evaluate engraftment, SCID mice were transplanted with 3x103, 6x103 and 12x103 non-cultured CD34+ cells or their entire progeny following 3-week expansion with cytokines only or cytokines + NA (n = 63). The frequency of SCID repopulating cells (SRC) was estimated by limiting dilution analysis as 1/ 36,756 (non-cultured), 1/19,982 (cytokines), 1/ 2,620 (NA) (SCID engraftment was considered as ≥0.5% human CD45+ cells). We found that, in correlation with homing, NA-treated cells have a 14- and 7.6-fold more SRC than non-cultured cells or cytokine-treated cells, respectively. The marked increase in SCID engraftment potential following culturing with NA may be attributed to both improved homing of CD34+ cells as well as higher proportion of early progenitor cells within the CD34+ cell compartment. Despite their numerical expansion, progenitor cells generated in cytokine-supplemented cultures have reduced homing and engraftment capacity. Our study demonstrates that NA modulates in-vitro expansion and augments the in-vivo homing and engraftment of CB-derived CD34+ cells cultured with cytokines.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

Ex Vivo Expansion of Frozen Cord Blood Products without Selection.

Blood ◽  
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.

Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 36-36
Author(s):  
Mehrnaz Safaee Talkhoncheh ◽  
Fredrik Ek ◽  
Aurelie Baudet ◽  
Christine Karlsson ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Expansion and Engraftment Potential of Cord Blood-Derived CD34+ Cells in NOD/SCID Mice

2006 ◽  
Vol 938 (1) ◽  
pp. 9-17 ◽  
Author(s):  
WILLEM E. FIBBE ◽  
WILLY A. NOORT ◽  
FRANK SCHIPPER ◽  
ROEL WILLEMZE
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Scid Mice ◽  
Cd34 Cells

scholarly journals Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4332-4332
Author(s):  
Xinxin Huang ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Lentiviral Vectors ◽  
Ex Vivo Expansion ◽  
Transcriptional Factor ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

Ex Vivo Proliferation and Differentiation of CD34+ Cells into Myeloid Progenitor Cells: Cord Blood Versus Mobilized Peripheral Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4175-4175
Author(s):  
Cecile De Bruyn ◽  
Alain Delforge ◽  
Marie-Christine Ngirabacu ◽  
Dominique Bron
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Myeloid Lineage ◽  
Myeloid Progenitor ◽  
Cd34 Cells ◽  
Myeloid Progenitor Cells ◽  
Mobilized Peripheral Blood

Abstract The purpose of our study was to evaluate the capacities of cord blood (CB) CD34+ cells to proliferate and differentiate ex vivo into myeloid lineage in response to cytokines and to compare them with mobilized peripheral blood (MPB) cells. Briefly, 2.5 × 104 CD34+ cells, isolated from CB (n=10) and MPB (n= 9), were cultured in 5 ml MacoBiotech HP01 (Macopharma) with SCF, Flt3-L, IL-3 and G-CSF. At day 9, 106 cultured cells were replated for 5 additional days. Cells were counted and evaluated for their CD34, CD13 and CD15 expression. Differentiation into myeloid compartment was assessed by CD11b and CD16 coexpression on CD15+ cells. We observed that leucocyte expansion was significantly higher in CB than in MPB at day 9 (24.3±3.8 vs 15.2±1.9) and at day 14 (224.7±54.2 vs 72.9±20.0). A similar difference was observed for CD34+ cell expansion (8.7±1.4 vs 3.4±0.5 at day 9 and 31.3±4.6 vs 7.6±2.4 at day 14). at day 9, despite superior CB leucocyte expansion, CD13+ and CD15+ cell number produced per CD34+ cell seeded at day 0 were similar in CB and in MPB (18.5±2.4 vs 14.4±2.5 for CD13+ and 7.1±1.3 vs 6.0±1.2 for CD15+). Increasing the culture period led to higher numbers of CD13+ and CD15+ cells in CB than in MPB. This increase was due to a total leucocyte expansion rather than to high CD13+ and CD15+ cell percentage. The distribution of CD11b−CD16−, CD11b+CD16− and CD11b+CD16+ subpopulations in CD15+ cells was comparable in CB and in MPB after 9 days of culture, with a majority of relatively immature CD11b−CD16− myeloid progenitor cells. However, after 5 additionnal days of culture, MPB CD15+ cells expressed a more mature phenotype than CB CD15+ cells, with a dramatic increase of CD11b+CD16− cells (promyelocytes and myelocytes). In conclusion, our study suggests that, despite the high CB cell capacity of expansion in our culture conditions, CB CD34+ cell differentiation process into myeloid lineage appears to be slower. This difficulty of CB cells to reach maturation in vitro is likely to be related with the longer delay of neutrophil recovery after CB transplantation.

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