Long-term Survival Of Embryonic Stem Cells In Adult Bone Marrow

Objective Embryonic stem cells (ESCs), derived from the inner cell mass of a blastocyst, possess two important characteristics:self-renewal and pluripotency toward all three embryonic germ layers. These remarkable characteristics make ESCs an attractive cell source in regenerative medicine. The important question regarding whether ESCs can survive in adult organs/tissues for the long-term remains unanswered. Bone marrow (BM) has been identified as a major source containing hematopoietic stem cells (HSCs) as well as non-hematopoietic stem cells, which include mesenchymal stem cells, multipotent adult progenitor cells, marrow-isolated adult multilineage inducible cells, and very small embryonic-like stem cells. Some of these non-hematopoietic bone-marrow-derived stem cells have been shown to have features in common with ESCs. Some express the Oct4 gene, and multipotent adult progenitor cells have been shown to differentiate into each of the three germ layer cells. However, the claim regarding the existence of ESC-like cells in adult tissues/organs such as bone marrow remains controversial largely due to the difficulty in isolating these extremely rare cells.In contrast to the previous studies that have examined this important yet unanswered question, we chose to approach it from the opposite direction by transplanting ESCs directly into the bone marrow to verify whether the bone marrow could provide a potential micro-environment for sustaining the pluripotent stem cells. A further rationale for carrying out our current study is the existing safety concerns regarding the potential of leftover or contaminated ESCs in therapeutic ESC products.Methods We approached this question by directly injecting mouse ESCs into syngeneic BM and then monitoring the survival and differentiation potential of the injected ESCs in BM based on the expression of GFP protein of the injected cells. To this end, an ESC line was derived from the Oct4-GFP transgenic mice in which GFP protein expression is driven by the Oct4 promoter, thereby indicating the status of pluripotency. As a control, an ESC line was also derived from the mice that constitutively express GFP that is driven by the chicken-β-Actin. The cells were injected via intra-tibia and then monitored at different time points after injection. The transplanted cells were visualized at several time points using two-photon microscopy to determine whether the bone marrow could sustain the pluripotent stem cells. Moreover, we further characterized the properties of the re-established cell lines recovered from the transplanted cells. To explore why the pluripotency of the re-derived cells are decreased, we compared the global gene-expression profiles of ESCs and the re-derived cell lines. To analyze the differentiation potential of ESCs after bone marrow transplantation, we examined the engrafted cells by flow cytometric analysis and the colony-forming unit-fibroblast assay.Results Despite the fact that some of the animals (28-29%in the irradiated hosts) developed tumors as expected, we intended to focus on the engrafted ESCs in the animals in which no tumor had occurred. We found that the Oct4+ cells could exist in recipient's BM for more than 100 days after transplantation as examined under the two-photon microscopy. Notably, most injected ESCs in BM differentiated into non-hematopoietic cells whereas few cells acquired hematopoietic cell surface markers. Interestingly, the engrafted cells were found in the contrary BM of the injected side as well as the blood circulation. We then harvested the Oct4+ cells in the BM at various time points after transplantation and attempted to re-establish the ESC lines. Eight ESC lines were recovered from a total of 141 irradiated recipients that were transplanted with the Oct4-driven GFP+ESCs. Those successfully recovered ESC lines from BM expressed known markers for embryonic pluripotency and were able to differentiate into three germ layers although germ line transmission is yet to be further defined. Gene expression profiling suggests time-dependent epigenetic alterations in the re-derived ESC lines as compared to their parental ESC line.Conclusions This study provides definitive evidence for long-term survival and differentiation of exogenous ESCs in adults. It has important implications for the understanding of stem cell development and maintenance, as well as for the safety evaluation of therapeutic ESC derivatives especially given the fact that rare un-differentiated ESCs can be contained in the final cellular products.