Human CD34⁺ Haematopoietic Stem Cells (HSC) from Umbilical Cord Blood Display an Endodermal Phenotype When Exposed to Activin A In Vitro.

Adult stem cells may not be tissue specific and transdifferentiation into cells committed to other tissues may occur. In vivo, bone marrow HSC can contribute to cells characteristic of resident endoderm-derived tissues, such as lung. We hypothesized that if transdifferentiation can occur, then in vitro evidence could be obtained from an enriched population of hematopoietic cells. This study evaluated whether endodermal lineages could be derived from human umbilical cord blood (UCB) cells in order to provide a novel source for the regeneration of non-hematopoietic tissues, such as the lung. UCB CD34⁺ hematopoietic stem cells were magnetically enriched to a purity greater than 92%. A fraction of these was immediately characterised by flow cytometry and the remaining cells were expanded for 3 days in serum-free media. The effects of a combination of growth factors on these cells, including FGF-4, HGF and Activin A, were evaluated after 15 and 30 days of culture. RT-PCR analysis revealed detectable mRNA levels from representative endodermal genes (SOX17, GATA4 and FOXa2) after 15 days of induction and epithelial genes cytokeratin (CK)-18 and E-cadherin (E-cad) after 30 days of induction. Control cells after 3 days expansion and freshly isolated CD34⁺ (day 0) showed detectable transcript levels of HOXb4, Gata1 and PECAM-1 but they were negative for all endoderm markers. A panel of endodermal and epithelial primary antibodies, GATA4, GATA6, E-cad, CK-18, Pan-CK and Albumin were immunolocalized in cultures after 15 and 30 days of induction showing gene and protein expression. In order to evaluate the temporal expression pattern of the endodermal genes excluding the endothelial cell component, enriched CD34⁺ cells were subsequently FACS-sorted to yield a CD34⁺CD45⁺ cell population of greater than 98% purity. Quantitative real time RT-PCR was used to measure fold changes in the expression of GATA4, FOXa2, SOX17, E-cad and CK-18 genes in response to low and high Activin A concentrations at 6 different time points of induction cell culture (0, 3, 5, 7, 9, 12 and 15 days). Exposure to Activin A increased the expression of the endoderm transcript levels as early as 3 days of induction for E-cad and SOX17 and at 7 and 9 days of induction for GATA4 and FOXa2, respectively. Data were normalized to the housekeeping gene ß-actin and were expressed relative to the level of the gene of interest at time zero. HSC cultivated without Activin A and evaluated at the same time points did not express endoderm transcripts. These findings show that in the presence of Activin A, hematopoietic stem cells can be induced to express genes and proteins indicative of endoderm lineage in vitro. Since Activin A is upregulated in damaged tissue and is required for wound healing, the data suggest a mechanism for in vitro “transdifferentiation” of HSC, especially in the repair of damaged organs, and indicate that this process may be applied therapeutically for regenerative medicine.