Membrane culture and reduced oxygen tension enhances cartilage matrix formation from equine cord blood mesenchymal stromal cells in vitro

Abstract Abstract 1443 Poster Board I-466 Background Hematopoietic stem cells (HSCs) are located mainly in the bone marrow interacting with a specific microenvironment called “stem cell niche”. The niche has been proven to be critical for stem cell regulation. Coculture with mesenchymal stromal cells (MSCs) has been used as an in vitro model to investigate the interaction between HSCs and MSCs. In our study we investigated the impact of normoxia and hypoxia on the distribution of HSC subsets with regard to their spatial localization in cocultures during ex-vivo expansion. Design and Methods Three HSC subsets are defined: (i) cells in supernatant (non-adherent cells); (ii) cells adhering on the MSC layer surface (phase-bright cells); (iii) cells beneath the MSC layer (phase-dim cells). Using pimonidazole binding we investigated the spatial distribution of hypoxic cells in various cell subsets. Cell cycle, cell division, immunophenotype and migratory capacity of the three HSC subsets under distinct oxygen tension were studied. In addition the impact of oxygen tension on HSCs via VEGF-A and SDF-1 were analyzed by ELISA and gene knockdown with siRNA. Results First we could show that phase-bright cells contained the highest proportion of cycling progenitors. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype. Next pimonidazole binding revealed that the most hypoxic area in the coculture is the compartment beneath MSC layer. Then we investigated the impact of hypoxia conditions on HSCs in cocultures. We could demonstrate that under hypoxic conditions phase-bright cells were significantly diminished and phase-dim cells were increased. Interestingly, the migratory capacity of phase-bright cells from cocultures performed under hypoxic conditions was consistently enhanced in comparison to normoxia (32.7 ±2.2.0% vs 17.6 ±2.6%, p<0.01). Surprisingly, the SDF-1 concentration was lower after hypoxic coculture (189 ±33μg/ml vs 352 ±40μg/ml, p<0.05). In contrast, the VEGF-A concentration was significantly increased compared to normoxic conditions (7.7 ±1.2ng/ml vs 4.5 ±1.0ng/ml, p<0.01). In addition we could demonstrate that the lower adhesion and higher migratory capacity of HSCs under hypoxia can be partially inversed by silencing VEGF-A with siRNA in MSCs. Conclusions Our data indicate that under our experimental conditions, the MSC surface is the dominant location where HSCs proliferate, whereas the compartment beneath the MSC layer seems to be a hypoxic niche dedicated to the maintenance of HSC stemness. The lower levels of SDF-1 in the supernatant may be explained by the increased internalization of SDF-1 by MSCs when cultured together with HSC. This hypothesis will require the concomitant analysis of protein and SDF-1 mRNA in MSC. In addition our data suggest that low oxygen tension facilitates HSC migration into the in-vitro niche provided by MSCs which preserves immaturity of HSCs and modifies the cytokine profile of MSCs. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Cord Blood Hematopoietic Progenitor Cell in Co-Culture with Bone Marrow Mesenchymal Stromal Cells: A Synergic Effect

Blood ◽

10.1182/blood.v118.21.4811.4811 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4811-4811

Author(s):

Camillo Almici ◽

Arabella Neva ◽

Rosanna Verardi ◽

Simona Braga ◽

Andrea Di Palma ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Progenitor Cell ◽

Cord Blood Sample ◽

Hematopoietic Stem

Abstract Abstract 4811 The number of hematopoietic stem and progenitor cells (HPCs) in cord blood units are limited and this can result in delayed engraftment. In vitro expansion of HPCs provides a perspective to overcome these limitations. Different combinations of cytokines as well as mesenchymal stromal cells (MSC) have been shown to separately support HPCs ex vivo expansion, but the combining effects are under evaluation. Data derived from ex vivo co-culture systems using MSC as a feeder layer suggest that cellular contacts could have a significant impact on expansion. We have evaluated the expansion rate of thawed cord blood samples (n=6) in a medium containing SCF (100 ng/ml) and G-CSF (100 ng/ml) plated over a pre-established bone marrow derived MSC layer in comparison to the absence of either MSC layer or cytokines. After 7 days cultures were demi-depopulated. At 14 days of culture adherent and non-adherent cells were harvested, counted and evaluated for antigens expression and clonogenic capacity. Immunophenotypic analysis was performed using CD34-PE, CD38-FITC, CD45-PE-Cy7, CD133-APC. Clonogenic assay was performed in semisolid methylcellulose culture medium (MethoCult, Stem Cell Technologies), CFU frequencies and total CFU numbers per cord blood sample were determined. After 14 days of culture, in the presence of MSC layer, an 11.2-fold increase (range 4.4–18.4) in total number of cells was observed, in comparison to a 4.8-fold increase (range1.1-10.35) in the absence of MSC layer. The presence of MSC layer generated a 4.3-fold increase (range 1.5–7.2) in the number of CD34 positive cells, compared to a 3.3-fold increase (range 0.9–5.7) in the absence of MSC; when considering the more immature CD34+/CD38− subpopulation the corresponding increase were 26.9-fold vs 2.85-fold, respectively. Moreover, the percentage of the CD34+/CD38− subpopulation was higher in the adherent compared to the non-adherent fraction (76% vs 15%). The selection effect given by the MSC layer was confirmed by the presence of hematopoiesis foci growing onto the MSC layer. Our data show that cord blood HPCs can be expanded in vitro, moreover the co-culture on a MSC layer shows a synergistic effect on TCN, CD34+ cells and on more primitive CD34+/CD38− cells. Therefore, a clinical protocol of cord blood HPCs and MSC co-culture could represent a promising approach for improving engraftment kinetics in cord blood transplant recipients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Wound-healing potential of human umbilical cord blood–derived mesenchymal stromal cells in vitro—a pilot study

Cytotherapy ◽

10.1016/j.jcyt.2015.06.011 ◽

2015 ◽

Vol 17 (11) ◽

pp. 1506-1513 ◽

Cited By ~ 14

Author(s):

Hi-Jin You ◽

Sik Namgoong ◽

Seung-Kyu Han ◽

Seong-Ho Jeong ◽

Eun-Sang Dhong ◽

...

Keyword(s):

Wound Healing ◽

Pilot Study ◽

Cord Blood ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Umbilical Cord Blood ◽

Stromal Cells ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord

Download Full-text

Oxygen Tension Strongly Influences Metabolic Parameters and the Release of Interleukin-6 of Human Amniotic Mesenchymal Stromal Cells In Vitro

Stem Cells International ◽

10.1155/2018/9502451 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Asmita Banerjee ◽

Andrea Lindenmair ◽

Ralf Steinborn ◽

Sergiu Dan Dumitrescu ◽

Simone Hennerbichler ◽

...

Keyword(s):

Oxygen Tension ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Human Amniotic Membrane ◽

Low Oxygen ◽

Inflammatory Parameters ◽

Short Time ◽

Amniotic Cells

The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. In vivo (in utero), cells of the hAM are exposed to low oxygen tension (1–4% oxygen), while the hAM is usually cultured in atmospheric, meaning high, oxygen tension (20% oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5% and 20% oxygen. We found 20% oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS); however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20% oxygen. To conclude, short-time cultivation at 20% oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy.

Download Full-text

Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood

Veterinary and Comparative Orthopaedics and Traumatology ◽

10.3415/vcot-08-10-0107 ◽

2009 ◽

Vol 22 (05) ◽

pp. 363-370 ◽

Cited By ~ 52

Author(s):

T. G. Koch ◽

T. Heerkens ◽

K. Besonov ◽

P. D. Thomsen ◽

D. H. Betts ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Umbilical Cord Blood ◽

Stromal Cells ◽

Supplementary Information ◽

Multiple Sources ◽

Chondrogenic Potential

Summary Objective: Orthopaedic injury is the most common cause of lost training days or premature retirement in the equine athlete. Cell-based therapies are a potential new treatment option in musculo-skeletal diseases. Mesenchymal stromal cells (MSC) have been derived from multiple sources in the horse including bone marrow and umbilical cord blood. The objective of this study was to provide an in vitro comparison of the chondrogenic potential in MSC derived from adult bone marrow (BM-MSC) and umbilical cord blood (CB-MSC). Results: MSC from both sources produced tissue with cartilage-like morphology that stained positive for proteoglycans and expressed cartilage markers. The CB-MSC pellets were larger and showed hyaline-like cartilage morphology as early as day six. Gene expression of collagen type 21, aggrecan and CD-RAP was higher in CB- than BM-MSC pel-lets. Expression of Sox9 mRNA was similar between CB- and BM-MSC pellets. Protein concentration of cartilage-derived retinoic acid sensitive protein was higher in culture medium from CB- than BM-MSC pellets. Conclusion: CB-MSC and BM-MSC were both capable of producing hyaline-like cartilage in vitro. However, in this study the MSC from umbilical cord blood appeared to have more chondrogenic potential than the BMMSC based on the cells tested and parameters measured.Supplementary Information for this paper is available on the VCOT website at www.VCOTonline.com.

Download Full-text