Expansion of Cord Blood Hematopoietic Stem Cells on the Amniotic Membrane Derived Mesenchymal Stem Cells

ABSTRACT: Because of insufficient number of umbilical cord blood hematopoietic stem cells (UCB-HSCs), expansion of these cells seems to be important for clinical application in adults. The aim of this study was to co-culture of UCB-HSCs with the amniotic membrane derived mesenchymal stem cells (AMMSCs) as a feeder layer in order to expand hematopoietic stem cells (HSCs). UCBs and amniotic membrane were collected from concern mothers. Ex vivo culture of UCB-HSCs were performed in four culture conditions: cytokine cocktail with MSCs feeder layer, cytokine cocktail, stem cell factor, and co-culture with MSCs without any cytokine. The number of total nucleated cells (TNC) was counted by hemocytometer. The HSC count and immunophenotyping of Mesenchymal stem cells (MSCs) and expanded HSC were evaluated by flow cytometry. Colony forming unit (CFU) assay was used to evaluate the potential of expanded HSCs for production of different lineage colonies. The mean fold changes of total nucleated cells (TNC) and CD34+ cells in the cytokine culture with feeder layer were higher than the cytokine culture without MSCs. However, in the co-culture system without cytokine, TNC and CD34+ cell numbers were increased up to 8 folds, but cell viability was more than 80% and differentiation rate was low. Our results demonstrated that we could increase the number of CD34+ cells of UCB that were used as primary HSC for transplantation.

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Gene-Chip Analysis of Cord Blood Derived CD34+ Cells Expanded on Bioartificial Materials.

Blood ◽

10.1182/blood.v104.11.4131.4131 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4131-4131

Author(s):

Joachim Oswald ◽

Christine Steudel ◽

Katrin Salchert ◽

Christian Thiede ◽

Gerhard Ehninger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Fibrillar Collagen ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Colony Forming Units

Abstract Expansion of hematopoietic stem cells from neonatal cord blood is an important issue for clinical uses since the number of CD34+ cells in individual cord blood samples is limited and often not sufficient for a successful engraftment in adult individuals. In vivo, hematopoietic stem cells reside in the bone marrow in close vicinity to stromal cells and extracellular matrix molecules. We have established a culture system for the ex vivo expansion of CD34+ cord blood cells utilizing fibrillar collagen 1 as a bioartificial matrix to enable cellular adhesion during cell culture. CD34+ hematopoietic stem cells were isolated via immunomagnetic separation from umbilical cord blood after informed consent and cultivated in presence of recombinant cytokines and reconstituted collagen 1 fibrils as matrix. After seven days of cultivation, expansion of cells, expression of surface molecules cells and expansion of colony forming units were assessed. Additionally gene expression profiling was performed with Affymetrix HG U133A chips interrogating 22,253 probe sets. As control, CD34+ cells were expanded in liquid culture without fibrillar collagen. The overall expansion of CD34+ cells was 4.2 fold + 1.7 compared to 11.1 fold + 2.9 for the control sample. The number of colony forming units (CFU) was increased in the collagen 1 containing samples was elevated (65.1 + 10.3 compared to 26.1 + 7.6 in the control). Gene expression analysis with chip technology showed up regulation of several cytokines (e.g. interleukin 8, interleukin 1a) and also of transcription factors with antiproliferative features like BTG2. The chip data have been verified with quantitative PCR using the Taqman technology. Our data support the idea that direct contact of CD34+ cells with fibrillar collagen 1 results in a delay in cell cycle progression which prevents a subsequent differentiation into more committed progenitors. Therefore fibrillar collagen 1 may serve as supportive matrix for the ex vivo expansion of cord blood derived CD34+ cells.

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

Blood ◽

10.1182/blood.v118.21.4810.4810 ◽

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.

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Mesenchymal stem cells from the Wharton's jelly of umbilical cord segments provide stromal support for the maintenance of cord blood hematopoietic stem cells during long-term ex vivo culture

Transfusion ◽

10.1111/j.1537-2995.2008.01926.x ◽

2008 ◽

Vol 48 (12) ◽

pp. 2638-2644 ◽

Cited By ~ 65

Author(s):

Tiki Bakhshi ◽

Ryan C. Zabriskie ◽

Shamanique Bodie ◽

Shannon Kidd ◽

Susan Ramin ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Stromal Support ◽

Ex Vivo Culture

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Preclinical Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v128.22.3859.3859 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3859-3859

Author(s):

Helen Fong ◽

Goar Mosoyan ◽

Ami Patel ◽

Ronald Hoffman ◽

Jay Tong ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Shelf Life ◽

Ex Vivo ◽

Culture Conditions ◽

Hematopoietic Stem ◽

Cell Product ◽

Cd34 Cells

Abstract Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34+ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients. Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34+ cells to generate an MK culture composed of defined MK populations: CD34+/CD41+/CD42b- MK precursors (MKP), immature CD34-/CD41+/CD42b- MK (iMK) and mature CD34-/CD41+/CD42b+ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34+ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34+ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41+ MKs obtained ranged from 8 to 33 MK per input CD34+ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34+ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x106 CD34+ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34+ cell would generate over 56x106 MK or the equivalent of 7x105 CD41+ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34+ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41+ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells from Human Umbilical Cord Blood

Cell Transplantation ◽

10.3727/000000007783465073 ◽

2007 ◽

Vol 16 (6) ◽

pp. 579-585 ◽

Cited By ~ 48

Author(s):

Guo-Ping Huang ◽

Zhi-Jun Pan ◽

Bing-Bing Jia ◽

Qiang Zheng ◽

Chun-Gang Xie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Cd34 Cells

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45–55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

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Mesenchymal stem cells promote a primitive phenotype CD34+c-kit+ in human cord blood-derived hematopoietic stem cells during ex vivo expansion

Cellular & Molecular Biology Letters ◽

10.2478/s11658-012-0036-1 ◽

2013 ◽

Vol 18 (1) ◽

Cited By ~ 13

Author(s):

Viviana Rodríguez-Pardo ◽

Jean Vernot

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Ex Vivo ◽

Cell Subpopulation ◽

Cell Phenotype ◽

Hematopoietic Stem ◽

Secreted Factors

AbstractThe purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increasedCD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.

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