The potential of umbilical cord blood multipotent stem cells for nonhematopoietic tissue and cell regeneration

Ontogeny of developmental hematopoiesis of human pluripotent stem cells.

Primary Isolates of multilineage progenitor cells (MLPC). (A) Initial isolates of MLPC. Freshly isolated MLPC and cells cultured less than 3 days exhibit a leukocyte-like morphology and a panel of key characteristic protein markers associated with these cells. (B) After morphologic conversion to fibroblastic phase (usually day 3 to day 7) MLPC attain their final culture-specific morphology and key characteristic protein phenotype. (C) and (F) Phase contrast of MLPC at culture day 3. (D) and (G), same cells seen in (C) and (F) CD34 (D) and CD45 (G) immunofluorescence. (E) and (H) Phase contrast (E) and CD45 immunofluorescence (H) of day 5 MLPC. The arrow in each photomicrograph points to a cell that has converted from the leukocyte phase to the fibroblast phase of morphology and has lost its expression of CD45 antigen. Cells that retain the leukocyte morphology and are positive for CD45 surround the fibroblastic stage cell. ^∗Arrows point to MLPC, which has attained fibroblastic morphology has lost expression of CD45, while the surrounding MLPCs in leukocyte morphology retain expression of CD45.

Comparison of multilineage progenitor cells (MLPC) to cord blood-derived mesenchymal stem cells (MSC) and bone marrow-derived MSC. MLPC and MSC cell lines were grown to 60% confluency in MSCGM medium (Cambrex). MLPC clone E8 was compared to BM-MSC (Cambrex) and cord blood-derived Clone A3 (BioE). While all cell types are typically positive for CD105, CD29, CD73, and CD44 the individual cell types could be clearly distinguished by examining CD9, CD90, and CD106. MLPC clones were positive for CD9 and CD106 while expressing a bright and dull population for CD90. The MSC lines were clearly negative for CD106, expressed a single bright population for CD90 and were either negative (BM-MSC) or dull (CB-MSC).

Comparative Expression of multilineage progenitor cells (MLPC) vs mesenchymal stem cells (MSC) mRNA by microarray. mRNA was obtained from logarithmically growing cultures of MLPC and bone marrow-derived MSC and subjected to competitive hybridization microarray utilizing a 942-gene array designed by Miltenyi to focus on stem cell–associated and lineage-commitment genes. Bars to the left of the zero axis are overexpressed by MSC, bars to the right of the zero axis are genes that are overexpressed by MLPC. Of the 942 genes analyzed by this array, 360 genes were expressed significantly different. MLPC were found to be relatively quiescent primitive cells that were capable of wide plasticity yet were not committed to lineage. MSC were found to be much more committed to stromal and connective tissue lineages overexpressing genes associated with extracellular matrix, growth factors, and proteins associated with bone and cartilage.

Multipotential differentiating capacity of multilineage progenitor cells (MLPC). Mesodermal differentiation: (A–D), Endodermal differentiation: (E) Ectodermal differentiation: (F–H). (A) Adipocytic differentiation. MLPC were grown for 10 days in the presence of adipogenic induction medium (Cambrex). Triglyceride-containing liposomes were visualized with Nile Red stain. (B) Osteocytic differentiation. MLPC were grown in the presence of Osteogenic Induction Medium (Cambrex) for 21 days. Osteoblasts and mineralization were visualized by staining with Alizarin Red. (C) Myogenic differentiation. MLPC were grown on fibronectin-coated chamber slides in the presence of Skeletal Muscle Growth Medium (Cambrex) supplemented with fibroblast growth factor (FGF) basic (100 ng/mL), 5′-azacytidine (10 μM, Sigma), and cardiogenol C (10 μM, Sigma) for 30 days. Cells were stained for myocyte-specific α-actinin. (D) Endothelial differentiation. MLPC are grown in the presence of Endothelial Growth Medium-Microvasculature (Cambrex) for 21 days. Cells were stained for endothelium-specific protein CD102. (E) Differentiation of hepatopancreatic precursor cells. MLPC were cultured for 30 days in the presence of Hepatocyte Complete Medium (Cambrex) supplemented with FGF basic (20 ng/mL), FGF-4 (20 ng/mL), and stem cell factor (SCF; 40 ng/mL). Three-dimensional bodies were stained for human proinsulin. (F,G,H). Neurogenic differentiation. MLPC were grown on laminin and poly-d-lysine–coated chamber slides in the presence of Neural Progenitor Maintenance Medium (Cambrex) supplemented with FGF-4 (20 ng/mL) for 30 days mixed cultures of neurons, astrocytes, and oligodendrocytes developed.