Cord Blood as a Very Valuable Source of Neonatal Cells but Embryonic-Like Nature Reevaluated
Authors: Buchheiser Anja , Liedtke Stefanie , Houben Amelie Pia , Waclawczyk Simon , Stephan Milaid , Radke Teja Falk , Wernet Peter , Koegler Gesine 
Published: April 2020

Abstract
Human umbilical cord blood has become a very valuable source for hematopoietic transplantation. Our group was able to show that CB contains non-hematopoietic stem cells, which were called unrestricted somatic stem cells (USSCs) with a multipotent differentiation potential. These cells have the potential to differentiate into different germ layers (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). Some studies have now reported a presumably embryonic like nature of cord blood cells. However, Nanog and Oct 4 harbours potential pitfalls for data misinterpretation due to pseudogenes and alternative spliced variants (Liedtke et al. 2007, Liedtke et al. 2008). The related data based on the stem cell markers Nanog and Oct4 concerning these results remain questionable. Therefore, we evaluated the embryonic-like nature of MNCs (n=7), USSC (n=7), CD34+ cells (n=7) derived from cord blood, MNCs from peripheral blood (n=3), MNCs (n=7) and MSCs (n=3) from bone marrow. Using RT-PCR, quantitative RT-PCR and immunohistochemistry, we studied the expression of the pluripotency markers Oct4, Nanog, Sox2 as well as the transcription factors Klf4 and cMyc utilized for the induction of pluripotent stem cells from adult human fibroblasts. The expression level of the transcription factors Klf4 and cMyc was nearly equal in all USSC cell lines and BM MSCs. We neither detected expression of Oct4, Nanog and Sox2 in all tested USSC cell lines nor in MNCs and CD34+ cells from cord blood nor in MSCs, MNCs and CD34+ cells from bone marrow. To increase the sensitivity of our method we performed quantitative Oct4 PCRs. This method revealed that USSCs reach the same Oct4 expression level as human dermal fibroblasts. These results are also supported by the inactive status of the telomerase. As a positive control we used the embryonic carcinoma cell line nTERA-2 showing a high expression of Oct4, Nanog and Sox2. In addition we were able to show that the markers SSEA1, SSEA3 and SSEA4 cannot be used as markers of an embryonic-like phenotype. SSEA-1 recognizes the CD15 epitope, SSEA-4 cross-reacts with an adult MSC subpopulation and SSEA3 was always negative applying the correct isotype controls. However, cord blood does not have to contain embryonic like cells, but it contains neonatal cells as USSC expressing Sox17. For hematopoietic stem cells (HSC) it had already been shown that the transcription factor Sox 17 is required to maintain fetal and neonatal HSC and distinguishes their transcriptional regulation from adult HSCs (Kim et al. 2007). Our results indicate that USSCs and cord blood are neonatal cells without expression of typical embryonic stem cell markers. In more than 10.000 unrelated Cord blood transplants performed so far, no tumor formation associated with an Oct4 positive cell/teratoma formation was observed. Therefore the embryonic-like nature of cord blood cells must be reconsidered.