In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway.
BACKGROUND
Pluripotent unrestricted somatic stem cells (USSC) from UC blood can differentiate into hepatic cells in the in utero sheep model, resulting in 20% human albumin-producing parenchymal hepatic cells without cell fusion or tumor-formation events. Additionally, we have shown in vitro differentiation of USSC by hepatocyte growth factor and oncostatin M induction, causing changes in the gene expression towards the endodermal lineage. Positive glycogen synthase expression and a positive periodic acid-schiff reaction demonstrated a functional production of polysaccharides in the cells.


METHODS
We describe the in vitro differentiation of USSC towards an endodermal pathway using different matrices, growth factors and organic substances. Also, co-cultures of USSC with primary cells of endodermal tissue were prepared to mimic the biologic niche. We investigated the effect of direct co-culture of USSC with primary rat hepatocytes or with sheep tissue of endodermal origin. Direct co-cultures were set up to ensure cell-cell contacts. For co-cultures without cell-cell contacts, transwell inlays with 1-microm membranes were used to separate the cells. Furthermore, the effect of endodermally conditioned medium was investigated. Changes in the gene expression patterns were analyzed by RT-PCR.


RESULTS
We have shown that USSC can differentiate in vitro into an endodermal-like cell with a phenotype similar to hepatic cells. Differentiation of USSC with growth factors, retinoic acid, matrigel matrix and different co-cultures led to an increased expression of albumin and also to the detection of GSC, SOX 17, Cyp2B6, Cyp3A4, Gys2, HNF4a, ISL-1 and Nkx6.1. In addition, functional albumin secretion was observed.


CONCLUSIONS
Although the differentiation assays demonstrated here produce only an immature hepatocyte-like cell, endodermaly differentiated USSC might be a useful alternative for cell replacement in the future.
Sensken
S
S
Institute for Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf Medical School, Duesseldorf, Germany.
Waclawczyk
S
S
Knaupp
A S
AS
Trapp
T
T
Enczmann
J
J
Wernet
P
P
Kogler
G
G
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D018920:Coculture Techniques; D003094:Collagen / Q000187:drug effects; D017077:Culture Media, Conditioned; D004338:Drug Combinations; D004707:Endoderm / Q000166:cytology* / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology* / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007797:Laminin / Q000187:drug effects; D053683:Oncostatin M / Q000494:pharmacology; D010503:Periodic Acid-Schiff Reaction; D011509:Proteoglycans / Q000187:drug effects; D051381:Rats; D012709:Serum Albumin / Q000378:metabolism; D012756:Sheep; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects; D014212:Tretinoin / Q000494:pharmacology
D017077:Culture Media, Conditioned; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D012709:Serum Albumin; D053683:Oncostatin M; C059485:matrigel; D014212:Tretinoin; D017228:Hepatocyte Growth Factor; D003094:Collagen