The Effect of IL-3 in Ex Vivo Expansion of UCB CD34+ Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4201-4201
Author(s):  
Jin-Young Paek ◽  
Do-Yeon Kim
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Ex Vivo ◽  
Expansion Ratio ◽  
Culture Conditions ◽  
Hematopoietic Stem ◽  
Cd 34 ◽  
Cd34 Cells ◽  
Cell Culture Conditions ◽  
Better Than

Abstract Background: Human umbilical cord blood (UCB) has been recognized as a source of hematopoietic stem cells for transplantation and the CD34+ cell dose is important factor predicting the hematologic recovery. However, the cell dose and CD34 (+) cells in one unit UCB are limited. This study compared the ex vivo culture conditions for expansion of total nucleated cells (TNC) and CD 34+ cells using isolated from UCB CD34 (+) and CD 133 (+) subset cells. Material and method: The isolation of CD34 (+) and CD133 (+) cells from UCB was done by AutoMACS and the purity was more than 95%. We set three different cell culture conditions in stroma-free, serum-free medium. The culture conditions were 1) TPO, stem cell factor (SCF), Flt3-1 2) TPO/SCF/FLT3-1 and IL 3 10ng/dl, 3) TPO/SCF/FLT3-1 and IL 6 10ng/dl. After 7 days and 14days culture, we have been evaluated TNC and CD 34 (+) cell number for expansion potential. Results: The TNC after 7 days and 14 days culture, in the CD 34(+) subset, the TPO/SCF/Flt3-1 condition was 15.6 fold and 8.0 fold and the TPO/SCF/Flt3-1 + IL-3 condition was 31.6 fold and 20.1 fold and the TPO/SCF/Flt3-1 + IL-6 condition was 25.5 fold and 12.0 fold, respectively. In the CD133 (+) subset, the TPO/SCF/Flt3-1 condition was 7.1 fold and 8.4 fold and in the TPO/SCF/Flt3-1 + IL-3 condition was 17.9 fold and 24.8 fold and in TPO/SCF/Flt3-1 + IL-6 condition was 15.9 fold and 19.8 fold, respectively. In terms of CD34 (+) cell population in the expanded cells, in the CD34 (+) subset, the TPO/SCF/Flt3-1 condition was 40%, the TPO/SCF/Flt3-1 + IL-3 condition was 64% and in the TPO/SCF/Flt3-1 + IL-6 condition was 40%, respectively. In the CD 133 (+) subset, the TPO/SCF/Flt3-1 condition was 83% and in TPO/SCF/Flt3-1 + IL-3 condition was 48% and in TPO/SCF/Flt3-1 + IL-6 condition was 62%, respectively. Conclusions: To obtain more CD 34(+) cell population among ex vivo expanded cells, 7 days culture was more effective than 14 days culture and CD 133(+) subset was better than CD 34(+) subset. In terms of cell expansion ratio TPO/SCF/Flt3-1 + IL-3 was better than TPO/SCF/Flt3-1 and TPO/SCF/Flt3-1+ IL-6, which would be support the relevance of 7 days culture and CD133 (+) subset and IL-3 for expansion of hematopoietic stem cell ex vivo expansion. Fig 1: The expansion ratio of both CD (34+)(a) and CD133 (+)(b) subset after 7 days and 14 days cultures in the 3 different cell culture conditions Fig 1:. The expansion ratio of both CD (34+)(a) and CD133 (+)(b) subset after 7 days and 14 days cultures in the 3 different cell culture conditions Fig 2: The CD34(+) cell percentages in the different culture conditions both CD34(+) and CD133(+) subsets. Fig 2:. The CD34(+) cell percentages in the different culture conditions both CD34(+) and CD133(+) subsets.

scholarly journals RNAi Screens for Maintenance of Hematopoietic Stem Cell Phenotype During Culture

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-45-SCI-45
Author(s):  
Jonas Larsson
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Cellular Stress Response ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stress Response Genes ◽  
Cd34 Cells ◽  
Epigenetic Regulators

Successful ex vivo expansion of hematopoietic stem cells (HSCs) could greatly enhance cell- and gene therapy applications in treatment of malignant and inherited hematological disorders. It has therefore been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs. We have developed RNAi screening strategies to identify modifiers of self-renewal/proliferation in human hematopoietic stem- and progenitor cells (HSCPs). Employing pooled lentiviral shRNA libraries targeted to human cord blood derived CD34+ cells, we use the limited persistence of HSPCs under ex vivo culture conditions as a basis for functional selection of shRNAs conferring prolonged maintenance or expansion of undifferentiated cells. From the screens we have been able to identify directly targetable genes such as p38 MAPK, which can be pharmacologically inhibited to enhance the stem cell output of cultured cord blood cells. Additionally, we have found several epigenetic regulators among the top-scoring genes, including genes of the cohesin family, which modulate chromatin architecture, and JARID2, an important modifier of polycomb repressive complex 2 (PRC2). Depletion of either the cohesin genes or JARID2 by shRNA impairs differentiation and enhances both in vitro expansion and the in vivo reconstitution capacity of human HSPCs. However, whether these genes can be targeted pharmacologically to support HSC expansion remains to be explored. The screens have further identified a number of shRNAs that display a remarkable ability to expand HSPCs in culture, but whose gene targets have not been validated, indicating that they are affecting one or more genes in a non-specific manner. We performed gene expression profiling of cells transduced with these off-target shRNAs to gain insight about the molecular context under which HSPCs are propagated ex vivo. A common expression signature was the down-modulation of cellular stress response genes, such as p38 MAPK (previously identified in the screens) as well as genes involved in NF-□B signaling. Indeed, we found that pharmacological inhibition of NF-□B signaling leads to a significant improvement of stem cell function, from ex vivo cultured cord blood derived CD34+ cells, as assessed by transplantation to NSG mice. The effect of NF-□B inhibition was most critical early during the culture where it reduced the levels of several inflammatory and stress-related cytokines that are induced as an immediate response to culture initiation. Taken together, findings from our RNAi screens indicate that ex vivo HSC expansion is facilitated by targeting two distinct gene categories: cellular stress response genes activated by the culture conditions, as well as epigenetic regulators controlling the balance between renewal and differentiation. In order to define the most optimal expansion conditions we are currently assessing the combined targeting of several of these factors in conjunction with other compounds such as SR1 and UM171 using cellular barcoding. Here, cells are genetically marked by lentivirally delivered barcode sequences prior to expansion culture, which enables a precise detection and comparison of the stem cell output from multiple different expansion conditions in a directly competitive manner within a single xenografted animal. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform

2019 ◽  
Vol 14 (3) ◽  
pp. 243-255 ◽  
Author(s):  
Glyn N Stacey ◽  
Peter W Andrews ◽  
Ivana Barbaric ◽  
Charlotta Boiers ◽  
Amit Chandra ◽  
...  
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Culture Conditions ◽  
Stem Cell Culture ◽  
Joint Workshop ◽  
Cell Culture Conditions

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.

scholarly journals Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1405-1412 ◽  
Author(s):  
DN Haylock ◽  
LB To ◽  
TL Dowse ◽  
CA Juttner ◽  
PJ Simmons
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells

Abstract Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage- CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.

Immobilized Thrombopoietin (TPO) Lipopeptide Mimic Supports Similar Signaling and CD34+ Cell Differentiation as Soluble TPO.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3150-3150
Author(s):  
Shara M. Dellatore ◽  
James A. King ◽  
Tor W. Jensen ◽  
Bi-Huang Hu ◽  
Phillip B. Messersmith ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Adhesion ◽  
Ex Vivo ◽  
Lipid Monolayer ◽  
Adherent Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Defined Culture

Abstract Ex vivo expansion of hematopoietic stem cells (HSCs) would greatly facilitate cell and gene therapies. However, HSC division in culture is associated with differentiation. This contrasts with sustained HSC expansion in vivo, and has led to the hypothesis that a stem cell niche supports self-renewal. It is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. We are developing a defined culture surface for the presentation of cytokines and cell adhesion molecule (CAM) ligands that are thought to be in the HSC niche. Peptide mimics of CAM ligands and cytokines conjugated to dipalmitoyl glycerol via a polyethylene glycol tether are incorporated into dipalmitoylphosphatidylcholine (DPPC) vesicles and deposited onto a hydrophobic surface to create a lipid monolayer. We have previously shown that this system effectively presents adhesive peptide ligands (Jensen et al., JACS 126:15223, 2004). The strategy for immobilizing lipopeptides has been extended to the presentation of a peptide mimetic for the hematopoietic growth factor thrombopoietin (TPO). The lipopeptide mimetic of TPO is based on the branched dimer mimic (TPOm) developed by Cwirla et al. (Science 276:1696, 1997). We have synthesized two versions of TPOm lipopeptide, the first linked to a lipid at both of the amine termini (TPOm-2L) and the second is linked by a single lipid at the carboxy terminus (TPOm-1L). This immobilization strategy does not interfere with the bioactivity of the TPOm as evidenced by cell adhesion and signaling assays. Adhesion was measured with a normal force assay at 30g using the TPO-responsive M07e cell line. We observed a dose-dependent increase in adhesion, with <5% adherent cells for DPPC surfaces and a plateau of ~70% adherent cells at 1.0 mol% TPOm-1L. There was much less adhesion to TPOm-2L (a maximum of ~25% adhesion). Selective adhesion to the TPOm lipopeptides persisted after 6 days of culture, both in the presence and absence of serum. Culture surfaces with TPOm lipopeptides elicit similar M07e cell signaling response kinetics via the ERK1,2 and STAT5 pathways as compared to soluble TPOm and recombinant human TPO (rhTPO). It is interesting that surface presentation of TPOm synergizes more extensively with stem cell factor (SCF) for the activation of STAT5 than does soluble TPOm. Experiments with bone marrow (BM) CD34+ cells show that surfaces incorporating TPOm-2L supplemented with SCF and flt-3 ligand (FL) support similar overall expansion and protection from apoptosis as controls of soluble TPOm or rhTPO with SCF and FL. Further, there was no difference in the ability of TPOm to retain CD34+ cells or CD34+Thy1+ cells. Also, BM CD34+ cell cultures supplemented with TPOm-1L alone supported similar megakaryocyte maturation, evidenced by the appearance of polyploid CD41+ cells after 9 and 12 days of culture, as those supplemented with soluble TPOm. An advantage of this presentation strategy is the potential to save on cytokines during long-term culture. Feeding cultures stimulated by TPOm lipopeptides requires only exchange of basal media. In summary, we have developed a method to present immobilized TPOm in an active conformation that supports cell adhesion and signaling as well as the expansion and differentiation of CD34+ cells.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

2016 ◽  
Vol 7 (2) ◽  
pp. 177-191 ◽  
Author(s):  
Javier Martin Gonzalez ◽  
Sophie M. Morgani ◽  
Robert A. Bone ◽  
Kasper Bonderup ◽  
Sahar Abelchian ◽  
...  
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Developmental Stages ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Stem Cell Culture ◽  
Cell Culture Conditions
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