Clonal and Quantitative In Vivo Assessment of Hematopoietic Stem Cell Differentiation Reveals Strong Erythroid Potential of Multipotent Cells.

Hematopoiesis is arguably one of the best understood stem cell systems; however, significant challenges remain to reach a consensus understanding of the lineage potential, heterogeneity, and relationships of hematopoietic stem and progenitor cell populations. To gain new insights, we performed quantitative analyses of mature cell production from hematopoietic stem cells (HSCs) and multiple hematopoietic progenitor populations. Assessment of the absolute numbers of mature cell types produced by each progenitor cell revealed a striking erythroid dominance of all myeloid-competent progenitors assessed, accompanied by strong platelet reconstitution. All populations with myeloid potential also produced robust numbers of red blood cells and platelets in vivo. Clonal analysis by single-cell transplantation and by spleen colony assays revealed that a significant fraction of HSCs and multipotent progenitors have multilineage potential at the single-cell level. These new insights prompt an erythroid-focused model of hematopoietic differentiation.

A Transient Developmental Hematopoietic Stem Cell Gives Rise to Innate-like B and T Cells.

The generation of distinct hematopoietic cell types, including tissue-resident immune cells, distinguishes fetal from adult hematopoiesis. However, the mechanisms underlying differential cell production to generate a layered immune system during hematopoietic development are unclear. Using an irreversible lineage-tracing model, we identify a definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients but does not persist into adulthood in situ. These HSCs are fully multipotent, yet they display both higher lymphoid cell production and greater capacity to generate innate-like B and T lymphocytes as compared to coexisting fetal HSCs and adult HSCs. Thus, these developmentally restricted HSCs (drHSCs) define the origin and generation of early lymphoid cells that play essential roles in establishing self-recognition and tolerance, with important implications for understanding autoimmune disease, allergy, and rejection of transplanted organs.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

NK Cells Preferentially Target Tumor Cells with a Cancer Stem Cell Phenotype.

Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24(+)/CD44(+), CD133(+), and aldehyde dehydrogenase(bright)) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.

Natural killer cell subsets differentially reject embryonic stem cells based on licensing.

BACKGROUND: Embryonic stem cells (ESC) and induced pluripotent stem cells provide great promise to the future of medicine. Because immune rejection represents a major obstacle to the success of all stem cell-based therapies, many recent studies have sought to determine the key immune mediators involved in ESC rejection. The role of natural killer (NK) cells and specifically the role of NK cell licensing is not well understood in ESC rejection. METHODS: Mouse or human ESCs were subjected to cytotoxicity assays involving their respective species-matched activated NK cells. Mouse ESCs were then transplanted to allogeneic recipients after depletion of NK cell subsets in the host. ESC engraftment was analyzed by bioluminescent imaging. RESULTS: Depletion of all NK cells in vivo resulted in the greatest amount of ESC engraftment, confirming a role for NK cells in ESC rejection. Importantly, depletion of the Ly49C/I or Ly49G2 NK cell subsets resulted in differential ESC engraftment and rejection. This indicates that NK cell rejection of allogeneic ESC is highly differential based on the presence of licensed NK cells. Blocking NKG2D in vitro resulted in less killing of mESC by allogeneic NK cells, indicating NKG2D is a likely mechanism for NK-mediated killing of mESC. CONCLUSIONS: In this study, we show that expression of inhibitory Ly49s correlates with the ability of NK cells to kill murine ESC in an NKG2D-dependent manner. This further suggests that the rejection of similar stem cell transplants in humans will be dependent upon the presence of licensed NK cells.