Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation.
Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.
Battula
Venkata Lokesh
VL
Department of Internal Medicine II, Division of Hematology, Immunology, Oncology and Rheumatology, University Clinic of Tübingen, Tübingen, Germany.
Bareiss
Petra M
PM
Treml
Sabrina
S
Conrad
Sabine
S
Albert
Ingrid
I
Hojak
Sigrid
S
Abele
Harald
H
Schewe
Bernhard
B
Just
Lothar
L
Skutella
Thomas
T
Bühring
Hans-Jörg
HJ
D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000951:Antigens, Neoplasm; D001854:Bone Marrow Cells / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D051157:Frizzled Receptors / Q000276:immunology / Q000378:metabolism*; D006028:Glycosphingolipids / Q000276:immunology / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010920:Placenta / Q000166:cytology*; D043562:Receptors, G-Protein-Coupled / Q000276:immunology / Q000378:metabolism*; D055746:Stage-Specific Embryonic Antigens
D015703:Antigens, CD; D000951:Antigens, Neoplasm; C432044:CDCP1 protein, human; D015815:Cell Adhesion Molecules; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; C121168:FZD10 protein, human; C105574:FZD3 protein, human; D051157:Frizzled Receptors; D006028:Glycosphingolipids; D018398:Homeodomain Proteins; D007381:Intermediate Filament Proteins; C486780:NANOG protein, human; C577484:NES protein, human; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D050814:Octamer Transcription Factor-3; D043562:Receptors, G-Protein-Coupled; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D016222:Fibroblast Growth Factor 2