Joachim Oswald

Mesenchymal Stem Cells Can Be Differentiated Into Endothelial Cells In Vitro

Human bone marrow‐derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes, chondrocytes, and adipocytes in vivo and in vitro. The aim of this study was to investigate the in vitro differentiation of MSCs into cells of the endothelial lineage. MSCs were generated out of mononuclear bone marrow cells from healthy donors separated by density gradient centrifugation. Cells were characterized by flow cytometry using a panel of monoclonal antibodies and were tested for their potential to differentiate along different mesenchymal lineages. Isolated MSCs were positive for the markers CD105, CD73, CD166, CD90, and CD44 and negative for typical hematopoietic and endothelial markers. They were able to differentiate into adipocytes and osteocytes after cultivation in respective media. Differentiation into endothelial‐like cells was induced by cultivation of confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor. Laser scanning cytometry analysis of the confluent cells in situ showed a strong increase of expression of endothelial‐specific markers like KDR and FLT‐1, and immunofluorescence analysis showed typical expression of the von Willebrand factor. The functional behavior of the differentiated cells was tested with an in vitro angiogenesis test kit where cells formed characteristic capillary‐like structures. We could show the differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells. These predifferentiated cells provide new options for engineering of artificial tissues based on autologous MSCs and vascularized engineered tissues.

Gene-Expression Profiling of CD34+ Hematopoietic Cells Expanded in a Collagen I Matrix

CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms‐like tyrosine kinase‐3 ligand, stem cell factor, and interleukin (IL)‐3. After 7 days of culture, the cell number, number of colony‐forming units (CFU‐C), and gene‐expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU‐C was greater than in control suspension cultures. Gene‐expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL‐8 and macrophage inhibitory protein 1α) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell‐cycle regulator BTG2/TIS21 and an inhibitor of the mitogen‐activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three‐dimensional collagen I matrix induces a qualitative change in the gene‐expression profile of cultivated HSCs.

Comparative analysis of proliferative potential and clonogenicity of MACS-immunomagnetic isolated CD34+ and CD133+ blood stem cells derived from a single donor

Abstract.  A novel stem cell marker prominin‐1 (CD133) has been shown to be expressed on a subpopulation of CD34+ haematopoietic stem and progenitor cells. The aim of this study was to compare in parallel commercially available CD34+ and CD133+ isolation methods based on paramagnetic bead‐coupled antibodies using clinical‐grade samples of mobilized peripheral blood from 10 individual healthy donors under identical conditions. The CD133 negative fraction from the first selection was used for CD34+ enrichment to obtain an additional CD34+/CD133− population. Although no significant difference in total cell expansion between cells isolated from the three procedures was observed in a 7‐day cytokine‐driven suspension culture, the long‐term culture‐initiating cell assay demonstrated that cells derived by CD34+ isolation contain less primitive progenitors than those isolated based on CD133+ selection. Interestingly, CD34+‐enriched progenitors, especially the CD34+/CD133− fraction, contained a significantly higher proportion of erythroid colony‐forming cells, whereas the highest content of myeloid colony‐forming cells was concentrated in the CD133+ selected cells. These subtle differences between CD34+ and CD133+ immunomagnetic selection will have to be explored for their potential clinical relevance.

Comparison of Flow Cytometry and Laser Scanning Cytometry for the Analysis of CD34 Hematopoietic Stem Cells

Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM). The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC. For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC.