A Wharton's Jelly Mesenchymal Stromal Cell Derived 3D Osteogenic Niche Allows for Cord Blood Stem Cell Expansion Using Cytokine-Free Culture Media

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4832-4832
Author(s):  
Omar S. Aljitawi ◽  
Peggy Keefe ◽  
Christianna Henderson ◽  
Salem Akel ◽  
Sunil Abhyankar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Liquid Culture ◽  
Conflicts Of Interest ◽  
Culture Media ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Wharton’S Jelly ◽  
Wharton's Jelly ◽  
Free Media

Abstract Abstract 4832 Introduction: In prior experiments, a 3 dimensional (3D) osteogenic niche was developed using Wharton's jelly mesenchymal stromal cells (WJMSC) that allowed for successful attachment of CD34 + umbilical cord blood (UCB) stem cells. The 3D osteogenic scaffold resulted in 10 times expansion of total nucleated cells (TNCs), however, most of the expanded cells were CD34 negative possibly secondary to their differentiation attributed to the cytokine-rich media. Accordingly, it was proposed to use a cytokine-free media in expansion of CD34+UCB using the same 3D osteogenic structure. Aims: To assess the ex vivo expansion of CD34+UCB stem cells in a 3D osteogenic niche using cytokine-free culture media. The expansion in 3D conditions was compared to 2D and liquid-culture conditions. Methods: CD34+ selected UCB stem cells were expanded in 3D osteogenic scaffold using cytokine-free media for 2 weeks. In 2D conditions, CD34+ UCB stem cells were expanded over a monolayer of osteogenic differentiated WJMSCs using the same cytokine-free media. In liquid culture conditions, the CD34+ UCB stem cells were expanded in culture flask. Pre- and post-expanded TNC was determined and post expansion CD34 expression was assessed using flow cytometry. Colony forming unit (CFU) assays were used to compare the expanded population in the three culture conditions. Results: TNCs were expanded 26X in 3D, 265X in 2D, and only 2X in liquid culture conditions. CD34 expressing cells increased 2.4X in 3D, 10X in 2D, and decreased in liquid cultures. Relative to 3D, CFU expansion in 2D was increased 2.6X. In 3D, CFUs were almost exclusively CFU-GM (>93%), compared to 2D (74%). Absence of CFU-E was noticed in the expanded population that remained attached to the 3D osteogenic scaffold. On the other hand, low CFU-E percentage was noticed in the population of expanded cells that dettached from the 3D osteogenic scaffold. Conclusions: The experimental conditions lead to a modest but significant expansion of CD34+UCB stem cells in 3D as well as 2D conditions. Though the majority of the expanded population did not express CD34, they retained their colonogenic potential. Contrary to the expanded cells that detached from the 3D osteogenic scaffold, the cells that remained attached did not form CFU-E. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse

Reproduction ◽  
2012 ◽  
Vol 143 (4) ◽  
pp. 455-468 ◽  
Author(s):  
Eleonora Iacono ◽  
Lara Brunori ◽  
Alessandro Pirrone ◽  
Pasquale Paolo Pagliaro ◽  
Francesca Ricci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Amniotic Fluid ◽  
Cord Blood ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Wharton’S Jelly ◽  
Blue Staining ◽  
Wharton's Jelly

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.

Usefulness of Wharton’s Jelly, Cord Blood, and Adipose Tissue as Alternative Sources of Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4250-4250
Author(s):  
Jun Ho Jang ◽  
Hyun Woo Lee ◽  
Young-Woo Eom ◽  
Seok Yun Kang ◽  
Joon Seong Park ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Cord Blood ◽  
Tissue Repair ◽  
Wharton’S Jelly ◽  
Population Doubling ◽  
Wharton's Jelly ◽  
Alternative Sources

Abstract Mesenchymal stem cells (MSCs) are a highly promising source of adult stem cells for purposes of cell therapy and tissue repair in the field of regenerative medicine. Although the most studied and accessible source of MSC is the bone marrow, the clinical use of bone marrow-derived MSCs (BMSCs) has presented problems, including pain, morbidity, and low cell number upon harvest. For those reasons, we isolated, cultured, and characterized MSCs from a number of tissues; including wharton’s jelly, cord blood, and adipose tissues that were discarded routinely in the past, and evaluated the usefulness of these MSCs compared to BMSCs. Proliferation ability of Wharton’s jelly-derived MSCs (WJ-MSCs), Cord blood-derived MSCs (CB-MSCs), or adipose tissue-derived MSCs (ASCs) was lost at passage 8–10 (22–27 population doubling), passage 7–10, or passage 7–12 (45–50 population doubling), respectively. WJ-MSCs, CB-MSCs, and ASCs expressed CD73, CD90, and CD105, CD90, CD105, and CD166, and CD44, CD73, CD90, and CD166, respectively, were absent for CD14, CD31, and CD45, and differentiated into osteoblast, adipocyte, and chondrogenic lineages under appropriate culture condition. In this study, like BMSCs, WJ-MSCs, CB-MSCs, and ASCs expressed similar cell surface antigens, were able to differentiate into mesenchymal lineages, and possessed highly proliferation potential. Therefore, MSCs isolated from wharton’s jelly, cord blood, and adipose tissue may become useful alternative sources of MSCs to cell therapy and tissue repair in the field of regenerative medicine.

scholarly journals Pressure Stimuli Improve the Proliferation of Wharton’s Jelly-Derived Mesenchymal Stem Cells under Hypoxic Culture Conditions

2020 ◽  
Vol 21 (19) ◽  
pp. 7092
Author(s):  
Sang Eon Park ◽  
Hyeongseop Kim ◽  
Soojin Kwon ◽  
Suk-joo Choi ◽  
Soo-young Oh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Primary Culture ◽  
Therapeutic Efficacy ◽  
Culture Conditions ◽  
Wharton’S Jelly ◽  
Wharton's Jelly ◽  
Accelerated Proliferation ◽  
Apoptotic Effects

Mesenchymal stem cells (MSCs) are safe, and they have good therapeutic efficacy through their paracrine action. However, long-term culture to produce sufficient MSCs for clinical use can result in side-effects, such as an inevitable senescence and the reduction of the therapeutic efficacy of the MSCs. In order to overcome this, the primary culture conditions of the MSCs can be modified to simulate the stem cells’ niche environment, resulting in accelerated proliferation, the achievement of the target production yield at earlier passages, and the improvement of the therapeutic efficacy. We exposed Wharton’s jelly-derived MSCs (WJ-MSCs) to pressure stimuli during the primary culture step. In order to evaluate the proliferation, stemness, and therapeutic efficacy of WJ-MSCs, image, genetic, and Western blot analyses were carried out. Compared with standard incubation culture conditions, the cell proliferation was significantly improved when the WJ-MSCs were exposed to pressure stimuli. However, the therapeutic efficacy (the promotion of cell proliferation and anti-apoptotic effects) and the stemness of the WJ-MSCs was maintained, regardless of the culture conditions. Exposure to pressure stimuli is a simple and efficient way to improve WJ-MSC proliferation without causing changes in stemness and therapeutic efficacy. In this way, clinical-grade WJ-MSCs can be produced rapidly and used for therapeutic applications.

scholarly journals Biomimetic microenvironmental preconditioning enhance neuroprotective properties of human mesenchymal stem cells derived from Wharton's Jelly (WJ-MSCs)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Wioletta Lech ◽  
Anna Sarnowska ◽  
Zuzanna Kuczynska ◽  
Filip Dabrowski ◽  
Anna Figiel-Dabrowska ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Neuroprotective Effect ◽  
Hippocampal Slices ◽  
Wharton’S Jelly ◽  
Trophic Factors ◽  
Differentiation Markers ◽  
Wharton's Jelly

Abstract Tuning stem cells microenvironment in vitro may influence their regenerative properties. In this study Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) were encapsulated in 3D hydrogels derived from human fibrin (FB) or platelet lysate (PL) and the oxygen level was adjusted to physiological normoxia (5% O2). The influence of the type of the scaffold and physiological normoxia conditions was tested on the WJ-MSCs' survivability, proliferation, migratory potential, the level of expression of selected trophic factors, cytokines, and neural markers. Encapsulated WJ-MSCs revealed high survivability, stable proliferation rate, and ability to migrate out of the hydrogel and the up-regulated expression of all tested factors, as well as the increased expression of neural differentiation markers. Physiological normoxia stimulated proliferation of encapsulated WJ-MSCs and significantly enhanced their neuronal, but not glial, differentiation. Ex vivo studies with indirect co-culture of organotypic hippocampal slices and cell-hydrogel bio-constructs revealed strong neuroprotective effect of WJ-MSCs against neuronal death in the CA1 region of the rat hippocampus. This effect was potentiated further by FB scaffolds under 5% O2 conditions. Our results indicating significant effect of oxygen and 3D cytoarchitecture suggest the urgent need for further optimization of the microenvironmental conditions to improve therapeutical competence of the WJ-MSCs population.

384 ISOLATION OF MESENCHYMAL STEM CELLS FROM WHARTON'S JELLY, CORD BLOOD, AND AMNIOTIC FLUID IN THE HORSE

2010 ◽  
Vol 22 (1) ◽  
pp. 348 ◽  
Author(s):  
E. Iacono ◽  
L. Brunori ◽  
A. Pirrone ◽  
B. Merlo
Keyword(s):  
Stem Cells ◽  
Amniotic Fluid ◽  
Cord Blood ◽  
Culture Medium ◽  
Wharton’S Jelly ◽  
Population Doubling ◽  
Blue Staining ◽  
Wharton's Jelly ◽  
Orthopedic Injuries ◽  
Alternative Sources

The effect of orthopedic injuries on the health of racehorse and racing financials can be dramatic. Bearing this in mind, there is naturally a great interest in new possible treatments for orthopedic injuries. In this study we evaluated equine amniotic fluid (AF), cord blood (CB), and Wharton’s jelly (WJ) as alternative sources of primitive, multipotent stem cells. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Samples were recovered at labor from 7 standardbred mares with ages between 6 and 15 years. Wharton’s jelly was incubated with collagenasis at 37°C for at least 2 hr. The resulting cell suspension was centrifuged, and the cell pellet was resuspended in culture medium (DMEM and TCM-199, 1:1) plus 10% FBS (GIBCO®, Invitrogen Corporation, Carlsbad, CA, USA), 100 IU mL-1 penicillin, and 100 μg mL-1 streptomycin). Amniotic fluid and CB were diluted 1:1 in PBS medium supplemented with antibiotic solution and centrifuged for 15 min at 300 x g. The pellet was resuspended in 5 mL of culture medium, placed on 5 mL of 70% Percoll solution, and centrifuged at 1200 x g for 30 min. The interface layer was then aspirated and resuspended in culture medium. All the cell lineages were washed seeded into 25 cm2 flasks and cultured in a 5% CO2 incubator at 38.5°C. Medium was refreshed after 48 h and then twice a week. Adherent fibroblast-like cells were tested for their differentiation potential at passage 3. Chondrogenic differentiation was performed using a micromass culture technique, whereas osteogenic differentiation was induced in monolayer culture (Mizuno H and Hyakusoku H 2003 J. Nippon Med. Sch. 70, 300-306). Evidence for differentiation was made via morphological, biochemical, and cytological evaluations. Sample volume was 15-55 mL for AF, 10-60 mL for CB, and 5-12.6 g for WJ. In 4 out of 6 (66.7%) AF, 5 out of 5 (100%) CB, and 6 out of 7 (85.7%) WJ samples, colonies with the classical mesenchymal stem cell (MSC) morphology of adherent fibroblastoid spindle-shaped cells growing in a monolayer were observed. Undifferentiated cells have been passaged up to 8 times, population-doubling times (DT) were calculated for a subset of cell passages, and data were analyzed by ANOVA (Statistica for Windows, Stat Soft Inc., Tulsa, OK, USA). In all samples, there were no significant differences (P > 0.05) between DT of all passages. The DT was greater (P < 0.05) forCB (2.6 ± 1.3 d) than for AF (2.1 ± 0.8 d) and WJ (1.9 ± 0.5 d). By passage 8, the cells had undergone 39.1 ± 1.2 cell-doubling numbers in AF, 35.1 ± 2.7 in CB, and 37.4 ± 2.0 in WJ. A spheroid structure and positive Alcian blue staining of matrix glycosaminoglycans illustrated chondrogenesis. Cellular morphology and positive von Kossa staining, as well as alkaline phosphatase activity, confirmed osteogenesis. Based on the results obtained, WJ, CB, and for the first time in the horse, AF represent alternative sources of stem cells. Their differentiation into osteocytes and chondrocytes demonstrated that equine MSC from these samples can be induced to form multiple cell types, which underlies their value for regenerative medicine in injured horses.

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.

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

Blood ◽  
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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