Animal transplantation models are essential to characterize the long-term in vivo engraftment capacity of putative hematopoietic stem cells derived from human embryonic stem cells (hESCs). We have previously demonstrated that hESCs can be routinely utilized to derive multiple hematopoietic cell lineages. Here, we use in vivo bioluminescence imaging (BLI) of stable luciferase (luc)-expressing hESCs to noninvasively monitor the dynamics of transplantation, engraftment, and growth of hESC-derived hematopoietic cells within individual animals over an extended time course. Luc expression under control of an EF1α promoter was introduced into the H1 hESC line using a self-inactivating lentiviral vector. Undifferentiated hESC colonies that stably expressed luciferase were established and selected, and the pluripotent capability of luc+ hESCs was first explored by teratoma formation. Undifferentiated luc+ human ES cells were intramuscularly injected into NOD/SCID mice. The dynamics of survival and growth of the hESCs was monitored by BLI using the IVIS Imaging System (Xenogen) at regular time points post-transplantation. There was a decrease of luminescent signal during the first 1–2 weeks. This was followed by a dramatic increase in luminescent signals after about 5 weeks, which correlated with teratoma size. Immunohistochemical analysis confirmed stable luc-expression in multiple differentiated cell types within the teratomas. We next used BLI to examine luc+ H1 hESCs that were induced to undergo hematopoietic differentiation by co-culture with S17 cells, to give rise to H1/S17 cells. Flow cytometric studies confirmed hematopoietic cells (CD34+, CD45+, CD31+, and c-kit+ cells) were derived from these differentiated luc+ hESCs, with 5–10% of H1/S17 cells being CD34+. Hematopoietic progenitors that gave rise to colonies of mature luc+ blood cells in a standard CFU assay were also observed from the H1/S17 cells. Luc-expression of differentiated hESCs was maintained at similar levels to those of the undifferentiated ES cells. To define the in vivo potential of luc+ hESC-derived hematopoietic cells, hESCs were allowed to differentiate on S17 cells for two weeks. SCID-repopulating cell studies were done by intravenous (iv) injection into sublethally irradiated NOD/SCID mice. After iv injection of 2–3 x106 unsorted luc+ H1/S17 cells, BLI showed the brightest signal in the lung at day 0 (within 2 hours), followed by a rapid decline in signal on the next day (day1). On day 8, most luc+ cells were detected in the abdomen and liver. Subsequently, after 6–12 weeks, multiple engraftment loci were identified in hematopoietic tissues. Flow cytometric analysis of bone marrow from these mice confirmed the presence of hESC-derived human CD45+ cells. Engraftment was also demonstrated after direct intra-bone marrow injection with as few as 60,000 CD34+ cells sorted from luc+ H1/S17 cells. Again, stable engraftment can be monitored by BLI for 8+ weeks. These results demonstrate that BLI has several important advantages as an effective non-invasive approach to track and quantitatively monitor in vivo engraftment of hematopoietic or other cell lineages derived from hESCs.

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