Search Results
Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells
2015, Reichert, Doreen, Friedrichs, Jens, Ritter, Steffi, Käubler, Theresa, Werner, Carsten, Bornhäuser, Martin, Corbeil, Denis
Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.
Distinguishing autocrine and paracrine signals in hematopoietic stem cell culture using a biofunctional microcavity platform
2016, Müller, Eike, Wang, Weijia, Qiao, Wenlian, Bornhäuser, Martin, Zandstra, Peter W., Werner, Carsten, Pompe, Tilo
Homeostasis of hematopoietic stem cells (HSC) in the mammalian bone marrow stem cell niche is regulated by signals of the local microenvironment. Besides juxtacrine, endocrine and metabolic cues, paracrine and autocrine signals are involved in controlling quiescence, proliferation and differentiation of HSC with strong implications on expansion and differentiation ex vivo as well as in vivo transplantation. Towards this aim, a cell culture analysis on a polymer microcavity carrier platform was combined with a partial least square analysis of a mechanistic model of cell proliferation. We could demonstrate the discrimination of specific autocrine and paracrine signals from soluble factors as stimulating and inhibitory effectors in hematopoietic stem and progenitor cell culture. From that we hypothesize autocrine signals to be predominantly involved in maintaining the quiescent state of HSC in single-cell niches and advocate our analysis platform as an unprecedented option for untangling convoluted signaling mechanisms in complex cell systems being it of juxtacrine, paracrine or autocrine origin.
Zebrafish In-Vivo Screening for Compounds Amplifying Hematopoietic Stem and Progenitor Cells: - Preclinical Validation in Human CD34+ Stem and Progenitor Cells
2017, Arulmozhivarman, Guruchandar, Kräter, Martin, Wobus, Manja, Friedrichs, Jens, Bejestani, Elham Pishali, Müller, Katrin, Lambert, Katrin, Alexopoulou, Dimitra, Dahl, Andreas, Stöter, Martin, Bickle, Marc, Shayegi, Nona, Hampe, Jochen, Stölzel, Friedrich, Brand, Michael, von Bonin, Malte, Bornhäuser, Martin
The identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34+ cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34+ cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.
Limbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro
2017, Fernández-Pérez, Julia, Binner, Marcus, Werner, Carsten, Bray, Laura J.
Limbal stromal cells (LSCs) from the human ocular surface display mesenchymal stromal cell characteristics in vitro. In this study, we isolated cells from the porcine limbal stroma (pLSCs), characterised them, and evaluated their ability to support angiogenesis and the culture of porcine limbal epithelial stem cells (pLESCs). The isolated cells adhered to plastic and grew in monolayers in vitro using serum-supplemented or serum-free medium. The pLSCs demonstrated expression of CD29, and cross-reactivity with anti-human CD45, CD90, CD105, CD146, and HLA-ABC. However, expression of CD105, CD146 and HLA-ABC reduced when cultured in serum-free medium. PLSCs did not undergo adipogenic or osteogenic differentiation, but differentiated towards the chondrogenic lineage. Isolated cells were also co-cultured with human umbilical vein endothelial cells (HUVECs) in star-shaped Poly(ethylene glycol) (starPEG)-heparin hydrogels to assess their pericyte capacity which supported angiogenesis networks of HUVECs. PLSCs supported the three dimensional HUVEC network for 7 days. The isolated cells were further growth-arrested and evaluated as feeder cells for pLESC expansion on silk fibroin membranes, as a potential carrier material for transplantation. PLSCs supported the growth of pLESCs comparably to murine 3T3 cells. In conclusion, although pLSCs were not completely comparable to their human counterpart, they display several mesenchymal-like characteristics in vitro.