Production of stem cells with embryonic characteristics from human umbilical cord blood.
When will embryonic stem cells reach the clinic? The answer is simple -- not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, - cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity--bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.
McGuckin
C P
CP
Stem Cell Therapy Programme, School of Life Sciences, Kingston University, Kingston upon Thames, Surrey, UK. c_mcgucken@hotmail.com
Forraz
N
N
Baradez
M-O
MO
Navran
S
S
Zhao
J
J
Urban
R
R
Tilton
R
R
Denner
L
L
D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; D015415:Biomarkers / Q000032:analysis; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D016207:Cytokines / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D006023:Glycoproteins / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D022781:Hepatocytes / Q000737:chemistry / Q000166:cytology; D006801:Humans; D050814:Octamer Transcription Factor-3; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; D013234:Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism
D000954:Antigens, Surface; D015295:Antigens, Tumor-Associated, Carbohydrate; D015415:Biomarkers; D016207:Cytokines; D004268:DNA-Binding Proteins; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D006028:Glycosphingolipids; D006133:Growth Substances; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; D014157:Transcription Factors; C052924:stage-specific embryonic antigen-3; C066584:stage-specific embryonic antigen-4