Generation of reconstituted hemato-lymphoid murine embryos by placental transplantation into embryos lacking HSCs

In order to increase the contribution of donor HSC cells, irradiation and DNA alkylating agents have been commonly used as experimental methods to eliminate HSCs for adult mice. But a technique of HSC deletion for mouse embryo for increase contribution of donor cells has not been published. Here, we established for the first time a procedure for placental HSC transplantation into E11.5 Runx1-deficient mice mated with G1-HRD-Runx1 transgenic mice (Runx1-/-::Tg mice) that have no HSCs in the fetal liver. Following the transplantation of fetal liver cells from mice (allogeneic) or rats (xenogeneic), high donor cell chimerism was observed in Runx1-/-::Tg embryos. Furthermore, chimerism analysis and colony assay data showed that donor fetal liver hematopoietic cells contributed to both white blood cells and red blood cells. Moreover, secondary transplantation into adult recipient mice indicated that the HSCs in rescued Runx1-/-::Tg embryos had normal abilities. These results suggest that mice lacking fetal liver HSCs are a powerful tool for hematopoiesis reconstruction during the embryonic stage and can potentially be used in basic research on HSCs or xenograft models.

Transcriptional profiling and single-cell chimerism analysis identifies human tissue resident T cells in the human skin after allogeneic stem cell transplantation

Tissue resident memory T cells (TRM) have recently emerged as crucial cellular players for host defense in a wide variety of tissues and barrier sites. Mouse models revealed that they are maintained long-term in loco unlike recirculating effector memory T cells (TEM). Insights into the maintenance and regulatory checkpoints of human tissue resident T cells (TRM) remain scarce, especially due to the obstacles in tracking T cells over time and system-wide in humans. We present a clinical model that allowed us to overcome these limitations. We demonstrate that allogeneic stem cell transplantation resulted in compartmentalization of host T cells in the human skin despite complete donor T cell chimerism in the blood, thus unmasking long-term persistence of tissue resident T cell subsets of host origin within the diverse skin T cell community. Single-cell transcriptional profiling paired with single-cell chimerism analysis provided an in-depth characterization of these bona fide skin resident T cells. Their phenotype, functions and regulatory checkpoints may serve therapeutic strategies for the treatment of autoimmune diseases and chronic infections, where their specific depletion versus maintenance, respectively, will have to be harnessed pharmaceutically.

Simultaneous Monitoring of Mutation and Chimerism Using Next-Generation Sequencing in Myelodysplastic Syndrome

Monitoring minimal residual disease (MRD) provides important information during treatment of hematologic malignancies. Chimerism analysis also provides key information after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recent advances in next-generation sequencing (NGS) have enabled identification of various mutations and quantification of mutant allele burden. In this study, we developed a new analytic algorithm to monitor chimerism applicable to NGS multi-gene panel in use to identify mutations of myelodysplastic syndrome (MDS). We enrolled patients who were diagnosed with MDS and received allo-HSCT and their corresponding donors. Monitoring MRD by NGS assay was performed using 53 DNA samples by calculating mutant allele burden after treatment. For monitoring chimerism by NGS, we selected 121 single nucleotide polymorphisms (SNPs) after careful stepwise evaluation and calculated average donor allele burden. Data obtained from NGS were compared with bone marrow findings, chromosome analysis and short tandem repeat (STR)-based chimerism. SNP-based NGS chimerism analysis was accurate and even superior to conventional STR method by overcoming the various technical limitations of STR. In addition, simultaneous monitoring of mutation and chimerism using NGS could implement comprehensive pre- and post-HSCT monitoring of various clinical conditions such as complete donor chimerism, persistent mixed chimerism, early relapse, and even donor cell-derived diseases.