The Wnt/β-catenin pathway is important for maintenance of the undifferentiated state of human embryonic stem cells (ESCs), hematopoietic stem cells (HSCs) and other stem cells. We here tested if β-catenin activation, by inhibiting GSK3 β with BIO (6-Bromoindirubin-3′-oxime), affects the differentiation status of murine multipotent adult progenitor cells (mMAPCs). mMAPCs were cultured with or without 0.1–2 μ M BIO, LIF, EGF and PDGF-BB, at low density (plating density of 100/cm2 and cells passed every 2 days) or at high confluency (like ESCs). Quantitative RT-PCR (Q-RT-PCR) was done weekly for the ESC-specific pluripotency transcripts, Oct4, Rex1, Nanog, UTF1 and E-Ras, and lineage commitment genes, vWF, VE-cadherin, Sox1, Nestin, CK19, Albumin, HNF3β and AFP. After 2–4 weeks, cells were analyzed by FACS for ESC/MAPC specific antigens, and by immunohistochemsitry for Oct4, β-catenin and E-cadherin protein. Furthermore, cells were differentiated into endothelial cells and hepatocytes by using VEGF-A and HGF/FGF4, respectively for 2 weeks. Q-RT-PCR was used to demonstrate lineage differentiation.

BIO caused a dose-dependent clustering of mMAPCs from 4 days after treatment at both cell densities. FACS phenotype of low- and high-density mMAPCs was not affected by BIO. Nanog mRNA was not detected in any of the cell populations. However, Oct4, Rex1, vWF, and CK19 mRNA levels decreased in a BIO dose-dependent manner in mMAPCs maintained at low density, while they acquired higher levels of Sox1 mRNA (P=NS). At high-density, Oct4 and Rex1 mRNA level decreased in a BIO dose-dependent manner for the first week. On the other hand, 0.1 μ M BIO-treatment induced higher levels of Oct4 and Rex1 in cells maintained at high density. β-catenin and E-cadherin protein was highly expressed in clusters of low-density mMAPCs induced by BIO as well as BIO-treated mMAPCs maintained at ESC densities. In both cell densities, only mMAPCs treated with 1 or 2 μM BIO contained large dense mMAPCs clusters that expressed very high levels of β-catenin and E-cadherin. There was no statistical difference of differentiation potential between BIO conditions and cell densities.

BIO may not allow culture of mMAPCs without loss of Oct4 as it did for ESCs, but induces clusters like ESCs in a BIO-dose dependent manner. High concentrations of BIO enhances β-catenin and E-cadherin protein expression. BIO may induce lineage commitment of mMAPCs. Despite loss of Oct4 mRNA and protein, MAPCs treated with BIO continue to have the ability to differentiate. Ongoing studies are testing whether BIO treated cells maintained at high density also maintain in vivo engraftment and differentiation potential.

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