scholarly journals Quiescence, Stemness and Adipogenic Differentiation Capacity in Human DLK1−/CD34+/CD24+ Adipose Stem/Progenitor Cells

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 214
Author(s):  
Florian M. Hatzmann ◽  
Asim Ejaz ◽  
G. Jan Wiegers ◽  
Markus Mandl ◽  
Camille Brucker ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Adipogenic Differentiation ◽  
Stromal Vascular Fraction ◽  
Quiescent State ◽  
Proliferation Markers ◽  
Differentiation Capacity ◽  
Cd34 Cells ◽  
Pluripotency Markers ◽  
Factor C

We explore the status of quiescence, stemness and adipogenic differentiation capacity in adipose stem/progenitor cells (ASCs) ex vivo, immediately after isolation from human subcutaneous white adipose tissue, by sorting the stromal vascular fraction into cell-surface DLK1+/CD34−, DLK1+/CD34dim and DLK1−/CD34+ cells. We demonstrate that DLK1−/CD34+ cells, the only population exhibiting proliferative and adipogenic capacity, express ex vivo the bonafide quiescence markers p21Cip1, p27Kip1 and p57Kip2 but neither proliferation markers nor the senescence marker p16Ink4a. The pluripotency markers NANOG, SOX2 and OCT4 are barely detectable in ex vivo ASCs while the somatic stemness factors, c-MYC and KLF4 and the early adipogenic factor C/EBPβ are highly expressed. Further sorting of ASCs into DLK1−/CD34+/CD24− and DLK1−/CD34+/CD24+ fractions shows that KLF4 and c-MYC are higher expressed in DLK1−/CD34+/CD24+ cells correlating with higher colony formation capacity and considerably lower adipogenic activity. Proliferation capacity is similar in both populations. Next, we show that ASCs routinely isolated by plastic-adherence are DLK1−/CD34+/CD24+. Intriguingly, CD24 knock-down in these cells reduces proliferation and adipogenesis. In conclusion, DLK1−/CD34+ ASCs in human sWAT exist in a quiescent state, express high levels of somatic stemness factors and the early adipogenic transcription factor C/EBPβ but senescence and pluripotency markers are barely detectable. Moreover, our data indicate that CD24 is necessary for adequate ASC proliferation and adipogenesis and that stemness is higher and adipogenic capacity lower in DLK1−/CD34+/CD24+ relative to DLK1−/CD34+/CD24− subpopulations.

scholarly journals Notch-Mediated Expansion of Human Hematopoietic Stem and Progenitor Cells By Culture Under Hypoxia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 28-29
Author(s):  
Daisuke Araki ◽  
Stefan Cordes ◽  
Fayaz Seifuddin ◽  
Luigi J. Alvarado ◽  
Mehdi Pirooznia ◽  
...  
Keyword(s):  
Er Stress ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Human Adult ◽  
Hypoxic Conditions ◽  
Cd34 Cells ◽  
Human Hscs ◽  
Ex Vivo Culture ◽  
Cells Cultured

Notch activation in human CD34+ hematopoietic stem/progenitor cells (HSPCs) by treatment with Delta1 ligand has enabled clinically relevant ex vivo expansion of short-term HSPCs. However, sustained engraftment of the expanded cells was not observed after transplantation, suggesting ineffective expansion of hematopoietic stem cells with long-term repopulating activity (LTR-HSCs). Recent studies have highlighted how increased proliferative demand in culture can trigger endoplasmic reticulum (ER) stress and impair HSC function. Here, we investigated whether ex vivo culture of HSPCs under hypoxia might limit cellular ER stress and thus offer a simple approach to preserve functional HSCs under high proliferative conditions, such as those promoted in culture with Delta1. Human adult mobilized CD34+ cells were cultured for 21 days under normoxia (21% O2) or hypoxia (2% O2) in vessels coated with optimized concentrations of Delta1. We observed enhanced progenitor cell activity within the CD34+ cell population treated with Delta1 in hypoxia, but the benefits provided by low-oxygen cultures were most notable in the primitive HSC compartment. At optimal coating densities of Delta1, the frequency of LTR-HSCs measured by limiting dilution analysis 16 weeks after transplantation into NSG mice was 4.9- and 4.2-fold higher in hypoxic cultures (1 in 1,586 CD34+ cells) compared with uncultured cells (1 in 7,706) and the normoxia group (1 in 5,090), respectively. Conversely, we observed no difference in expression of the homing CXCR4 receptor between cells cultured under normoxic and hypoxic conditions, indicating that hypoxia increased the absolute numbers of LTR-HSCs but not their homing potential after transplantation. To corroborate these findings molecularly, we performed transcriptomic analyses and found significant upregulation of a distinct HSC gene expression signature in cells cultured with Delta1 in hypoxia (Fig. A). Collectively, these data show that hypoxia supports a superior ex vivo expansion of human HSCs with LTR activity compared with normoxia at optimized densities of Delta1. To clarify how hypoxia improved Notch-mediated expansion of LTR-HSCs, we performed scRNA-seq of CD34+ cells treated with Delta1 under normoxic or hypoxic conditions. We identified 6 distinct clusters (clusters 0 to 5) in dimension-reduction (UMAP) analysis, with a comparable distribution of cells per cluster between normoxic and hypoxic cultures. Most clusters could be computationally assigned to a defined hematopoietic subpopulation, including progenitor cells (clusters 0 to 4) and a single transcriptionally defined HSC population (cluster 5). To assess the relative impact of normoxia and hypoxia on the HSC compartment, we performed gene set enrichment analysis (GSEA) of cells within HSC cluster 5 from each culture condition. A total of 32 genes were differentially expressed, and pathways indicative of cellular ER stress (unfolded protein response [UPR], heat shock protein [HSP] and chaperone) were significantly downregulated in hypoxia-treated cells relative to normoxic cultures (Fig. B). When examining expression of cluster 5 top differentially expressed genes across all cell clusters, we observed a more prominent upregulation of these genes within transcriptionally defined HSCs exposed to normoxia relative to more mature progenitors (Fig. C, red plots). Hypoxia lessened the cellular stress response in both progenitors and HSCs, but the mitigation was more apparent in the HSC population (Fig. C, grey plots), and decreased apoptosis was observed only within the HSC-enriched cluster 5 (Fig. D). These findings are consistent with several reports indicating that HSCs are more vulnerable to strong ER stress than downstream progenitors due to their lower protein folding capacity. In conclusion, we provide evidence that ex vivo culture of human adult CD34+ cells under hypoxic conditions enables a superior Delta1-mediated expansion of hematopoietic cells with LTR activity compared with normoxic cultures. Our data suggest a two-pronged mechanism by which optimal ectopic activation of Notch signaling in human HSCs promotes their self-renewal, and culture under hypoxia mitigates ER stress triggered by the increased proliferative demand, resulting in enhanced survival of expanding HSCs. This clinically feasible approach may be useful to improve outcomes of cellular therapeutics. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effect of local anesthetics on viability and differentiation of various adult stem/progenitor cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Young Hoon Kim ◽  
Ga Young Park ◽  
Nechama Rabinovitch ◽  
Solaiman Tarafder ◽  
Chang H. Lee
Keyword(s):  
Progenitor Cells ◽  
Local Anesthetics ◽  
Adipogenic Differentiation ◽  
Cell Types ◽  
Dose Effect ◽  
Specific Gene ◽  
Gene Expressions ◽  
Differentiation Capacity ◽  
Matrix Deposition ◽  
Lineage Differentiation

Abstract Background Local anesthetics (LAs) are widely used to control pain during various clinical treatments. One of the side effects of LAs, cytotoxicity, has been investigated in various cells including stem/progenitor cells. However, our understanding of the effects of LAs on the differentiation capacity of stem/progenitor cells still remains limited. Therefore, a comparative study was conducted to investigate the effects of multiple LAs on viability and multi-lineage differentiation of stem/progenitor cells that originated from various adult tissues. Method Multiple types of stem/progenitor cells, including bone marrow mesenchymal stem/progenitor cells (MSCs), dental pulp stem/progenitor cells (DPSCs), periodontal ligament stem/progenitor cells (PDLSCs), and tendon-derived stem/progenitor cells, were either obtained from a commercial provider or isolated from adult human donors. Lidocaine (LD) and bupivacaine (BP) at various doses (1×, 0.75×, 0.5×, and 0.25× of each physiological dose) were applied to the different stem/progenitor cells for an hour, followed by induction of fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation. Live/dead and MTT assays were performed at 24 h after the LD or BP treatment. At 2 weeks, qRT-PCR was conducted to evaluate the gene expressions associated with differentiation. After 4 weeks, multiple biochemical staining was performed to evaluate matrix deposition. Results At 24 h after LD or BP treatment, 1× and 0.75× physiological doses of LD and BP showed significant cytotoxicity in all the tested adult stem/progenitor cells. At 0.5×, BP resulted in higher viability than the same dose LD, with variance between cell types. Overall, the gene expressions associated with fibrogenic, chondrogenic, osteogenic, and adipogenic differentiation were attenuated in LD or BP pre-treated stem/progenitor cells, with notable dose-effect and dependence on types. In contrast, certain doses of LD and/or BP were found to increase specific gene expression, depending on the cell types. Conclusion Our data suggest that LAs such as LD and BP affect not only the viability but also the differentiation capacity of adult stem/progenitor cells from various anatomical sites. This study sheds light on stem cell applications for tissue regeneration in which isolation and transplantation of stem cells frequently involve LA administration.

scholarly journals The Effect of Early Rounds of ex vivo Expansion and Cryopreservation on the Adipogenic Differentiation Capacity of Adipose-Derived Stromal/Stem Cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
C. Durandt ◽  
C. Dessels ◽  
C. da Silva ◽  
C. Murdoch ◽  
M. S. Pepper
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Adipogenic Differentiation ◽  
Isolated Cells ◽  
Differentiation Potential ◽  
Differentiation Capacity ◽  
Surface Marker Expression ◽  
Cd36 Expression ◽  
The Impact ◽  
Stromal Stem Cells

Abstract Multipotent adipose-derived stromal/stem cells (ASCs) are candidates for use in cellular therapies for the treatment of a variety of conditions/diseases. Ex vivo expansion of freshly isolated ASCs may be necessary prior to clinical application to ensure that clinically relevant cell numbers are administered during treatment. In addition, cryopreserving cells at early passages allows for storage of freshly isolated cells for extended periods of time before expanding these cells for clinical usage. There are however several concerns that these laboratory-based procedures may alter the characteristics of the cells and in so doing decrease their regenerative potential. In this study we report on the impact of early rounds of cryopreservation (P0) and ex vivo expansion (P0 to P5) on the phenotypic characteristics and adipogenic differentiation potential of ASCs. Our results show that ASCs that upregulate CD36 expression during adipogenic differentiation gradually decrease with increasing expansion rounds. The consequent decrease in adipogenic differentiation capacity was evident in both gene expression and flow cytometry-based phenotypic studies. Successive rounds of expansion did not however alter cell surface marker expression of the cells. We also show that early cryopreservation of ASCs (at P0) does not affect the adipogenic differentiation potential of the cells.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Environmental Cues Can Promote Symmetrical Division of Functional Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1397-1397
Author(s):  
Nadim Mahmud ◽  
Kazumi Yoshinaga ◽  
Craig Beam ◽  
Hiroto Araki
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Widespread clinical use of ex-vivo expanded human umbilical cord blood (CB) grafts has been limited by lack of proper understanding of factors regulating self-renewal type of symmetric cell divisions. The expansion of the number of functional hematopoietic stem cells (HSC) ex-vivo requires the creation of an environment which favors symmetrical division. In our current studies, addition of late acting cytokines, (GM-CSF, IL-6, Epo) with early acting cytokines (thrombopoietin, SCF, Flt-3 ligand) resulted in loss of expansion of stem/progenitor cells. These data indicate that modification of HSC fate is not fully independent of external humoral influences. We have previously demonstrated that following treatment of CD34+ cells with 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA) there is a 10- fold increase in the number of SCID mouse repopulating cells (SRC). This increase of SRC, however, occurred concomitantly with an increase in absolute number of CD34+CD90+ cells as well as primitive progenitors which gives rise to colony forming unit Mix lineage (CFU-Mix). We hypothesized that if the primary CD34+ cells generates CFU-Mix/CFU-GM in a ratio of ‘X’, then to observe a higher rate of symmetric cell division we would expect to see the ratio increased (>X) in the 5azaD/TSA treated cells in comparison to cells cultured in the absence of 5azaD/TSA (< X). Interestingly, analyses of our data suggest that when 5azaD/TSA treated CD34+ cells are cultured for 5 days and assayed for colonies we observed a significant increase in the ratio of CFU-Mix/CFU-GM in contrast to cells cultured in cytokines alone, 0.373 ± 0.06 and 0.066 ± 0.032 respectively. The ratio of CFU-Mix/CFU-GM of CB CD34+ cells (day 0) was 0.262 ± 0.045. These findings indicate that 5azaD/TSA treatment promotes the ratio of CFU-Mix/CFU-GM possibly by enhancing symmetric division of CFU-Mix while in the absence of 5azaD/TSA treatment the culture condition likely induces differentiation. In addition, we have also investigated the ratio of progenitor cells/differentiated cells by assessing the ratio of human CD34+ cells/CD33+ cells in the bone marrow of immunodeficient mice following transplantation (8 weeks) of equal numbers of CD34+ cells. The ratio of CD34+ cells/CD33+ cells following transplantation of 5azaD/TSA treated cells was 0.52 ± 0.14 (n = 11) while in the absence of 5azaD/TSA the ratio dropped to 0.31± 0.16 (n = 4). The ratio following transplantation of primary CD34+ (day 0) cells was 0.62 ± 0.14 (n = 6). These data suggest that 5azaD/TSA treated cells maintain the balance of generation of CD34+ cells/CD33+ cells at a comparable rate to that of primary CD34+ cells, while the CD34+ cells generated in the absence of 5azaD/TSA promotes generation of more differentiated cells. Alternatively, it is also possible that 5azaD/TSA treatment of CD34+ cells in the culture results in inhibition of myeloid differentiation at the cost of proliferation. However, the latter possibility is unlikely, since treatment of CB cells with 5azaD/TSA results in an increase in the absolute number of progenitors including SRC possessing both myeloid and lymphoid differentiation potential. Taken together, these data support our hypothesis that chromatin modifying agents in the culture is capable of promoting self-renewal type of symmetric cell division possessing in vivo multilineage marrow repopulating potential.

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 340-340
Author(s):  
Pratima Chaurasia ◽  
Dmitriy Berenzon ◽  
Ronald Hoffman
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase Inhibitors ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Significant Proportion ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Cell Product ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Abstract 340 Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB. The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%). We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone. We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populations Histone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells. The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4. We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures: No relevant conflicts of interest to declare.

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 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.

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