Planarian immobilization, partial irradiation, and tissue transplantation.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed. Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration. Both tissue transplantation and partial irradiation have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

Amputation induces stem cell mobilization to sites of injury during planarian regeneration.

How adult stem cell populations are recruited for tissue renewal and repair is a fundamental question of biology. Mobilization of stem cells out of their niches followed by correct migration and differentiation at a site of tissue turnover or injury are important requirements for proper tissue maintenance and regeneration. However, we understand little about the mechanisms that control this process, possibly because the best studied vertebrate adult stem cell systems are not readily amenable to in vivo observation. Furthermore, few clear examples of the recruitment of fully potent stem cells, compared with limited progenitors, are known. Here, we show that planarian stem cells directionally migrate to amputation sites during regeneration. We also show that during tissue homeostasis they are stationary. Our study not only uncovers the existence of specific recruitment mechanisms elicited by amputation, but also sets the stage for the systematic characterization of evolutionarily conserved stem cell regulatory processes likely to inform stem cell function and dysfunction in higher organisms, including humans.

Mouse models of hematopoietic engraftment: limitations of transgenic green fluorescent protein strains and a high-performance liquid chromatography approach to analysis of erythroid chimerism.

Transgenic mouse strains ubiquitously expressing green fluorescent protein (GFP) have enabled investigators to develop in vivo transplant models that can detect donor contributions to many different tissues. However, most GFP transgenics lack expression of the reporter in the erythroid lineage. We evaluated expression of GFP in the bone marrow of the OsbY01 transgenic mouse (B6-GFP) in the context of CD71 and TER-119 expression and found that GFP fluorescence is lost prior to the basophilic erythroblast stage of development. However, platelets in B6-GFP mice were found to be uniformly positive for GFP. We therefore used the GFP transgenic model in combination with allelic variants of CD45 and the hemoglobin beta (Hbb) chain to develop a model system that allows all blood lineages to be followed in a mouse model of bone marrow transplantation (BMT). To detect Hbb variant molecules, we developed a new protocol based on high-performance liquid chromatography that is sensitive and precise, allowing rapid and quantitative analysis of erythroid chimerism. Platelet and leukocyte engraftment were detected by flow cytometry. BMT into sublethally irradiated (4 Gy) recipients demonstrated the failure of B6-GFP-derived cells to engraft relative to B6-CD45(a)-derived cells, suggesting that an immune barrier may prevent efficient engraftment of the transgenic cells in a setting of minimal ablation. These results establish limitations in the use of transgenic GFP expression as a donor marker in transplantation models.