scholarly journals Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Yun Gyeong Kang ◽  
Jee-Yeong Jeong ◽  
Tae-Hee Lee ◽  
Ho Sup Lee ◽  
Jung-Woog Shin
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Hydrostatic Pressure ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Physical Conditions ◽  
Clonogenic Potential ◽  
Nonadherent Cells

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems.

Ex-Vivo Expansion of Multipotent CD133+ Stem Cells with VEGF, FLT3l and SCGF.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4147-4147
Author(s):  
Sonja Loges ◽  
Martin Butzal ◽  
Uta Fischer ◽  
Ursula M. Gehling ◽  
Dieter K. Hossfeld ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Facs Analysis ◽  
Hematopoietic Stem ◽  
Large Numbers

Abstract The rare CD133+ stem cell population contains both hematopoietic and endothelial progenitors. Successful ex-vivo expansion of this multipotent population would therefore be of great benefit in many clinical settings including stem cell transplantation and gene therapy. We developed a cell culture system containing the recombinant human cytokines vascular endothelial growth factor (VEGF), FLT3 ligand (FLT3L) and stem cell growth factor (SCGF) for ex-vivo expansion of purified human CD133+ stem cells obtained from leukapheresis products from patients pre-treated with G-CSF. FACS analysis, colony assays and NOD-SCID transplantation studies were performed to monitor stem cell and endothelial phenotype in-vitro and in-vivo. Cultivation with VEGF, FLT3L and SCGF induced a mean 2200-fold increase of total cell counts in 5 weeks. FACS analysis revealed persistence of 6–15% CD133+ stem cells indicating proliferation and survival of primitive hematopoietic stem cells. 5–6% of the proliferating cells expressed the endothelial markers CD144 (VE-Cadherin) and von-Willebrand factor (vWF). Ex-vivo expanded stem cells could be differentiated into adherent endothelial cells after withdrawal of SCGF and FLT3L allowing generation of large numbers of endothelial cells. Colony-assays showed an increase of hematopoietic and endothelial colonies after 5 weeks of ex-vivo expansion indicating simultaneous proliferation of hematopoietic and endothelial precursors under the established culture conditions (CFU-E 60-fold, CFU-GEMM 48-fold, CFU-GM 59-fold, CFU-G 99-fold, CFU-M 1356-fold and CFU-EC 1843-fold). To assess in-vivo functionality, hematopoietic stem cells expanded ex-vivo for 7, 14, 21 and 32 days were transplanted into sublethally irradiated NOD-SCID mice. For each expansion period, the mean percentage of anti-human CD45 positive bone marrow cells 3 months post-transplantation was 11, 3, 3 and 1%, respectively. Human CD45+ cells for each set of experiments contained a mean of 15, 26, 8 and 32% T-cells (CD3+), 9, 0, 7 and 21% B-cells (CD19+), 24, 2, 2 and 11% monocytes (CD14+), 21, 3, 1 and 12% granulocytes (CD33+) and 19, 37, 44 and 24% stem cells (CD34+) (d7 (n=5), d14 (n=4), d21 (n=7) and d32 (n=6) respectively). Our experiments showed multilineage engraftment of human stem cells expanded for more than 4 weeks ex-vivo. Therefore our culture system provides a tool to generate large numbers of human stem and endothelial cells for clinical purposes.

Recombinant DEK Enhances Ex Vivo expansion of Human Cord Blood and Mouse Bone Marrow Hematopoietic Stem Cells in a CXCR2- and Hspg-Dependent Manner

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 779-779
Author(s):  
Maegan L. Capitano ◽  
Nirit Mor-Vaknin ◽  
Maureen Legendre ◽  
Scott Cooper ◽  
David Markovitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Inhibitory Function

Abstract DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin remodeling, and mRNA processing. Endogenous DEK regulates hematopoiesis, as BM from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Moreover, recombinant mouse (rm) DEK inhibits HPC colony formation in vitro. We now show that rmDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers (~2 fold) and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being more sensitive than control mice to this suppression. In contrast, in vivo administration of rmDEK to wild type and DEK-/- mice enhanced numbers of phenotypic LT-HSC. This suggests that DEK may enhance HSC numbers by blocking production of HPCs. We thus assessed effects of DEK on ex vivo expansion of human CD34+ cord blood (CB) and mouse Lin- BM cells stimulated with SCF, Flt3 ligand, and TPO. DEK significantly enhanced ex vivo expansion of rigorously-defined HSC by ~3 fold both on day 4 (~15 fold increase from day 0) and 7 (~29 fold increase from day 0) when compared to cells expanded without DEK. Expanding HSC with DEK also resulted in a decrease in the percentage of apoptotic HSC. Further studies were done to better define how DEK works on HSC and HPC. As extracellular DEK can bind to heparan sulfate proteoglycans (HSPG), become internalized, and then remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 673; Saha et al., 2013, PNAS, 110: 6847), we assessed effects of DEK on the heterochromatin marker H3K9He3 in the nucleus of purified mouse lineage negative, Sca-1 positive, c-Kit positive (LSK) BM cells by imaging flow cytometry. DEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK cells, indicating that rmDEK can be internalized by LSK cells and mediate heterochromatin formation. We also investigated whether inhibiting DEK's ability to bind to HSPG would block the inhibitory function of DEK in HPC. Blocking the synthesis of, the surface expression of, and the binding capability of HSPG blocked the inhibitory effect of DEK on colony formation. Blocking the ability of DEK to bind to HSPG also blocks the expansion of HSC in ex vivo expansion assays, suggesting that DEK mediates its function in both HSC and HPC by binding to HSPG but with opposing effects. To further evaluate the biological role of rmDEK, we utilized single-stranded anti-DEK aptamers that inactivate its function. These aptamers, but not their control, neutralized the inhibitory effect of rmDEK on HPC colony formation. Moreover, treating BM cells in vitro with truncated rmDEK created by incubating DEK with the enzyme DPP4 (DEK has targeted truncation sites for DPP4) eliminated the inhibitory effects of DEK, suggesting that DEK must be in its full- length form in order to perform its function. Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, and as DEK is a chemoattractant for mature white blood cells, we hypothesized that DEK may manifest at least some of its actions through CXCR2, the receptor known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case, we first confirmed expression of CXCR2 on the surface of HSC and HPC and then determined if neutralizing CXCR2 could block DEK's inhibitory function in HPC. BM treated in vitro with rmDEK, rhIL-8, or rmMIP-2 inhibited colony formation; pretreating BM with neutralizing CXCR2 antibodies blocked the inhibitory effect of these proteins. DEK inhibition of CFU-GM colony formation is dependent on Gai-protein-coupled receptor signaling as determined through the use of pertussis toxin, which is a mechanism unique to DEK, as we have previously reported that IL-8 and MIP-1a are insensitive to the inhibitory effects of pertussis toxin. Blocking the ability of DEK to bind to CXCR2 also inhibited the expansion of HSC in an ex vivo expansion assay. This suggests that DEK binds to CXCR2, HSPG or both to mediate its function on HPC and HSC, enhancing HSC but decreasing HPC numbers. Therefore, DEK may be a crucial regulatory determinant of HSC/HPC function and fate decision that is utilized to enhance ex vivo expansion of HSC. Disclosures No relevant conflicts of interest to declare.

Osteogenic- and Adipogenic- Differentiated Bone Marrow-Derived Mesenchymal Stem Cells Fail to Support Expansion of Adult Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4810-4810
Author(s):  
Olga Kulemina ◽  
Izida Minullina ◽  
Sergey Anisimov ◽  
Renata Dmitrieva ◽  
Andrey Zaritskey
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Feeder Layers

Abstract Abstract 4810 Ex vivo expansion and manipulation of primitive hematopoietic cells has become a major goal in the experimental hematology, because of its potential relevance in the development of therapeutic strategies aimed at treating a diverse group of hematologic disorders. Osteoblasts, mesenchymal stem/progenitor cells (MSC/MPC), adipocytes, reticular cells, endothelial cells and other stromal cells, have been implicated in regulation of HSC maintenance in endosteal and perivascular niches. These niches facilitate the signaling networks that control the balance between self-renewal and differentiation. In the present study, we evaluated and compared the effects of three different stromal feeder layers on expansion of HSPC derived from BM and cord blood (CB): BM mesenchymal stem cells (MSC), osteoblast-differentiated BM mesenchymal stem cells (Ost-MSC) and adipocyte-differentiated BM mesenchymal stem cells (Ad-MSC). BM-MSC cultures were established from plastic adherent BM cell fractions and analyzed for immunophenotype, frequency of colony forming units (CFU-F), frequency of osteo- (CFU-Ost) and adipo- (CFU-Ad) lineage progenitors. Cultures with similar clonogenity (CFU-F: 26,4 ± 4,5%) and progenitors frequency (CFU-Ost: 14,7 ± 4,5%; CFU-Ad: 13,3 ± 4,5%) were selected for co-culture experiments. All MSC were positive for stromal cell-associated markers (CD105, CD90, CD166, CD73) and negative for hematopoietic lineage cells markers (CD34, CD19, CD14, CD45). CD34+ cells were separared from BM and CB samples by magnetic cell sorting (MACS) and analyzed for CD34, CD38 and CD45 expression. Feeder layers (MSC, Ost-MSC, Ad-MSC) were prepared in 24-well plates prior to co-culture experiments: MSCs (4×104 cells/well) were cultured for 24 h and either used for following experiments or stimulated to differentiate into either osteoblasts or adipoctes according to standard protocols. CD34+ cells (3500-10000 cells per well) were co-cultured in Stem Span media with or without a feeder layers and in the presence of cytokines (10 ng/mL Flt3-L, 10 ng/mL SCF, 10ng/mL IL-7) for 7 days. Expanded cells were analyzed for CD34, CD38 and CD45 expression. Results are shown on figures 1 and 2. As expected, CB-derived HSPC expanded much more effectively than BM-derived HSPC. The similar levels of expansion were observed for both, the total number of HSPC, and more primitive CD34+CD38- fraction in the presence of all three feeder layers. Ost-MSC supported CB-derived HSPC slightly better than MSC and Ad-MSC which is in a good agreement with data from literature (Mishima et.al., European Journal of Haematology, 2010), but difference was not statistically significant. In contrast, whereas BM-MSC feeder facilitated CD34+CD38- fraction in BM-derived HSPC, Adipocyte-differentiated MSC and osteoblast-differentiated MSC failed to support BM-derived CD34+CD38- expansion (11,4 ±.4 folds for MSC vs 0,9 ±.0,14 for Ad-MSC, n=5, p<0,01 and 0,92 ±.0,1 for Ost-MSC, n=5, p<0,01).Figure 1.Cord Blood HSPC ex vivo expansionFigure 1. Cord Blood HSPC ex vivo expansionFigure 2.Bone Marrow HSPC ex vivo expansionFigure 2. Bone Marrow HSPC ex vivo expansion Conclusion: BM- and CB-derived CD34+CD38- cells differ in their dependence of bone marrow stroma. Coctail of growth factors facilitate CB HSPC expansion irrespective of lineage differentiation of supporting MSC feeder layer. In contrast, primitive BM CD34+CD38- HSPC were able to expand only on not differentiated MSC. Disclosures: No relevant conflicts of interest to declare.

Effective ex vivo expansion of hematopoietic stem cells using osteoblast-differentiated mesenchymal stem cells is CXCL12 dependent

2010 ◽  
Vol 84 (6) ◽  
pp. 538-546 ◽  
Author(s):  
Seiji Mishima ◽  
Atsushi Nagai ◽  
Sk Abdullah ◽  
Chikashi Matsuda ◽  
Takeshi Taketani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

Expansion of Cord Blood Stem Cells Mediated by Epigenetic Mechanisms Remain Permissive to External Environmental Cues

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3868-3868
Author(s):  
Hiroto Araki ◽  
Kazumi Yoshinaga ◽  
Sudhakar Baluchamy ◽  
Benjamin Petro ◽  
Donald Lavelle ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Trichostatin A ◽  
Ex Vivo Expansion ◽  
Division Rate ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Cytokine Combination

Abstract Widespread use of ex vivo expanded hematopoietic stem cells (HSC) has been largely limited by the lack of proper understanding of factors regulating symmetric self-renewing HSC divisions. We have previously reported that the addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase inhibitor, trichostatin A (TSA) in the culture is capable of expanding cord blood (CB) HSC as detected by in vivo SCID repopulating cells (SRC) assay in immunodeficient mice. The increase in SRC during ex vivo expansion culture was associated with greater transcript and protein products of genes implicated in HSC self-renewal (Araki et al. Blood 2007). In order to determine whether variation of exogenous cytokine cocktails added in the culture influences the degree of expansion of HSC treated with 5azaD/TSA, we have cultured CD34+ CB cells in the presence of various cytokine combinations. Interestingly, despite treatment of CB cells with 5azaD/TSA the expansion of stem/progenitor cells varied greatly, depending on the combinations of cytokines used in the culture, ranging between 5 to 12 fold differance. The cytokine combination containing stem cell factor (SCF), Flt3-ligand (FL) and thrombopoietin (TPO) was found to promote maximal expansion of primitive CD34+CD90+ cells following treatment with 5azaD/TSA in comparison to other cytokine combinations used (GM-CSF+SCF+IL-3+IL-6+EPO, SCF+FL+TPO+IL-3, SCF+FL+TPO+IL-6, SCF+FL+TPO+IL-3+IL-6, SCF+IL-3+IL-6). Our results also indicate the importance of sequential addition of 5azaD followed by TSA for the net expansion of HSC. Reversal of the sequence of addition of 5azaD and TSA (TSA followed by 5azaD) resulted in almost complete abrogation of the expansion of primitive CD34+CD90+ cells, and this loss of expansion corresponded with decreased acetylation of histone H4. We have further demonstrated that despite pre-treatment with sequential 5azaD/TSA, various cytokine cocktails in the culture can affect the rate of CD34+CD90+ cell divisions which influences both in vitro clonogenic potential and in vivo SRC potential. The higher in vivo hematopoietic engraftment potential of 5azaD/TSA treated cells in the presence of the optimal cytokine combination (SCF+FL+TPO) is likely due to expansion of a relatively primitive HSC population in the culture which divides slower than the cells expanded in the presence of other cytokine combinations (i.e. SCF+FL+TPO+IL-3+IL-6). Further studies will be needed to understand the molecular mechanism of the loss of functional potential depending on culture conditions. Thus far, in a transwell culture system, CD34+CD90+ cells that have been expanded with 5azaD/TSA show greater migration potential towards stroma derived factor (SDF-1) than CD34+CD90+ cells that have been expanded in cytokines alone without 5azaD/TSA treatment. Most importantly the fraction of migrating cells present in the 5azaD/TSA treated expanded culture was comparable to unmanipulated primary CB CD34+ cells, a likely factor contributing to better engraftment in an immunodeficient mouse model. Our current studies indicate that HSC remain responsive to external humoral influences even after treatment with chromatin modifying agents. The relatively slower cell division rate of CB cells in the presence of 5azaD/TSA might be a critical determinant for the retention of HSC functional capability following ex vivo expansion.

The impact of parathyroid hormone treated mesenchymal stem cells on ex-vivo expansion of cord blood hematopoietic stem cells

Gene Reports ◽  
2019 ◽  
Vol 17 ◽  
pp. 100490
Author(s):  
Mohammadhosein Esmaeili ◽  
Vahid Niazi ◽  
Ali Akbar Pourfathollah ◽  
Mir Kamran Mousavi Hosseini ◽  
Mozhdeh Nakhlestani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Parathyroid Hormone ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
The Impact

Pyrimido-Indole Derivatives Are Novel Agonists of Human Cord Blood Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 650-650
Author(s):  
Iman Fares ◽  
Jalila Chagaroui ◽  
Yves Gareau ◽  
Stéphane Gingras ◽  
Nadine Mayotte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Fed Batch ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The widespread use of cord blood (CB) unit in transplantation is limited with low number of long-term hematopoietic stem cells (LT-HSCs) and progenitors. Several approaches have been developed to expand HSC ex vivo such as automated and continuous medium delivery (fed-batch), notch delta ligand and SR1 (antagonist of aryl hydrocarbon receptor (AhR)). Concurrent with these studies, we hypothesized that small molecule with potent LT-HSC stimulating activity might be identified and potentiated in fed-batch culture system. Accordingly, we tested a library of more than 5000 small molecules for their in vitro expansion of CD34+CD45RA- cells. Most of the identified hits, except one (UM729) synthesized in our institute, suppress AhR pathway. Structure activity relationship was performed on UM729 to generate a more potent analog named UM171. This optimized molecule was 10-20 times more potent with an effective concentration of 15-20 nM when tested for its ability to expand CD34+CD45RA- cells. When compared to SR1, UM171 delivered in a fed-batch system for 12 and 16 days showed a better expansion of HSC phenotypes and lower apoptotic cell number compared to SR1 or DMSO controls. Also, UM171-expaned cultures showed higher number in multipotent progenitors (CFU-GEMM) and long term initiating cells (LTC-IC) compared to DMSO controls. Further studies showed the UM171 did not affect division rate, and its effect in expanding HSC phenotype was reversible. When combined with SR1, UM171 showed a better suppression of differentiation and led to a higher CFU-GEMM expansion compared to the single treatment of the compounds or DMOS controls. These observations suggest that UM171+SR1 cooperate to enhance ex vivo expansion of progenitor cells and suppress differentiation. To determine the in vivo activity of the expanded CD34+ CB cells, we transplanted fresh (un-manipulated) and 12-day cultured cells in NSG mice and monitored the human hematopoietic reconstitution after 20 and 30 weeks post-transplantation. Frequencies of day0 equivalent LT-HSCs were 13-fold higher in UM171 expanded cultures compared to fresh or fed-batch cultures supplemented with DMSO or SR1. Secondary experiments indicated that UM171 ex vivo treatment did not appear to affect the capability of LT-HSC to expand in primary recipients and hence similarly reconstituted secondary animals for at least 18 more weeks. This suggests that UM171 expands LT-HSC ex vivo without losing their engraftment potential. To further investigate UM171 mechanism of action, RNA- Seq expression profiling was performed. Unlike SR1 or DMSO controls, UM171 treatment was accompanied by a marked suppression of transcripts associated with erythroid and megakaryocytic differentiation and up-regulation of membrane protein transcripts such as EPCR and TEMEM 183a. In summery, UM171 is the first molecule identified so far that enables a robust ex vivo expansion of human CD34+ CB cells that sustain their in vivo activity independent of AhR suppression. Conversely, AhR suppression was limited to expand cells with less durable self-renewal potential. This study could enhance the use of small yet well HLA-matched CB units to become a prioritized source for stem cells transplantation. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document