Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos

The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W^sh/W^sh), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse–human chimeric platform allows for a new approach to study NC development and diseases.Genetically engineered mice have been highly informative in studying the developmental origin of many inherited diseases (1–3). However, mouse models often fail to reproduce the pathophysiology of human disorders due to interspecies divergence, such as metabolic differences between mouse and human (4), or differences in genetic background (5). To overcome some of the limitations of transgenic mouse models, transplantation of disease-relevant human cells into mice has been informative and is frequently used in cancer research. However, this approach is primarily restricted to the study of end-stage-disease cell types and provides only limited insight into tumor initiation and early progression of the disease under in vivo conditions, with the exception of the hematopoietic lineages, where human hematopoietic stem cells were found to successfully engraft into immune-deficient mice and provided a powerful approach for studying blood diseases (6).Somatic cell reprogramming provides patient-specific induced pluripotent stem cells (iPSCs) that carry all genetic alterations contributing to the disease pathophysiology and thus allows for generating the disease-relevant cell types in culture (7). However, many complex diseases involve progressive cellular or genetic alterations that occur before the manifestation of a clinical phenotype. Therefore, it is not clear whether a disease-relevant phenotype can be observed in short-term cultures of cells derived from patients with long-latency diseases, such as Parkinson''s or Alzheimer’s disease or cancers like melanoma. A major challenge is establishing model systems that, using human embryonic stem cells (hESCs) or hiPSCs, will allow for the investigation of human disease under appropriate in vivo conditions.Transplantation of hiPSCs or hiPSC-derived cells into mouse embryos would present an attractive solution to many of the aforementioned limitations. The main advantage of such an approach is that the transplanted cells would integrate into the embryo and participate in normal embryonic development, and consequently could be studied over the lifetime of the mouse. Currently, it is controversial whether the injection of hESCs/hiPSCs into preimplantation mouse blastocysts can generate even low-grade chimeric embryos (8–11). As an alternative approach, we explored whether multipotent somatic cells would be able to functionally integrate into postgastrulation mouse embryos and allow for the generation of mouse–human chimeras. We investigated the potential of human neural crest cells (hNCCs), derived from hESCs/hiPSCs, to integrate into the mouse embryo and contribute to the NC-associated melanocyte lineage. The NC, a multipotent cell population, arises at the boundary between the neuroepithelium and the prospective epidermis of the developing embryo. Trunk NCCs migrate over long distances, with the lateral migrating NCCs generating all of the melanocytic cells of the animal’s skin (12).NCC migration, development, and differentiation into various tissues have been studied in vivo by generating quail–chick NC chimeras. In this model, donor quail tissues were grafted into similar regions of developing chicken embryos (13). The experimental approach of our present study was based on the generation of mouse–mouse NC chimeras that had been created by injection of primary mouse NCCs into the amniotic cavity of embryonic-day (E) 8.5 embryos (14, 15). The donor mouse NCCs (mNCCs), having been placed outside of the embryo, enter into the neural tube, presumably through the still-open neural pores, and transverse the epidermis. The donor mNCCs used in this previous study were collected from pigmented C57BL/6 mice, whereas the host embryos were derived from BALB/c albino mice. Thus, contribution of the donor mNCCs to the host embryo could be determined by the presence of pigmentation in the coats of the injected mice. The injected primary mNCCs contributed to coat color formation in the head and hind limb regions only, but not in the midtrunk area, likely reflecting the entry point of the cells through the neural pores with the anterior–posterior movement of the cells being hindered by endogenous melanoblasts (15). Indeed, when embryos carrying the white-spotted c-Kit mutation (W^sh/W^sh), which lack melanoblasts, were used as a host, extensive coat color contribution revealing anterior–posterior cell migration was observed, presumably because the donor NCCs could spread into the empty niches (14).Here, we differentiated mouse, rat, and human ESCs or iPSCs into NCCs that were injected in utero into E8.5 albino wild-type and c-Kit–mutant W^sh/W^sh embryos. Both the mouse and human NCCs migrated laterally under the epidermis and ventrally into deeper regions of the embryo. Importantly, analysis of postnatal animals derived from mouse, rat, or human NCC-injected embryos displayed coat color pigmentation from the donor cells. Our results demonstrate that NCCs from different species can integrate into the developing mouse embryo, migrate through the dermis, and differentiate into functional pigment cells in postnatal mice. The generation of postnatal mouse–human chimeras carrying differentiated and functional human cells allows for a novel experimental system in which to study human diseases in an in vivo, developmentally relevant environment.