The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we ...demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.
Stem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to ...recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (approximately 10⁶ kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases.
Adipose tissue‐derived multipotent stromal cells (AT‐MSCs) are studied as an alternative to bone marrow‐derived multipotent stromal cells (BM‐MSCs) for immunomodulatory treatment. In this study, we ...systematically compared the immunomodulatory capacities of BM‐MSCs and AT‐MSCs derived from age‐matched donors. We found that BM‐MSCs and AT‐MSCs share a similar immunophenotype and capacity for in vitro multilineage differentiation. BM‐MSCs and AT‐MSCs showed comparable immunomodulatory effects as they were both able to suppress proliferation of stimulated peripheral blood mononuclear cells and to inhibit differentiation of monocyte‐derived immature dendritic cells. However, at equal cell numbers, the AT‐MSCs showed more potent immunomodulatory effects in both assays as compared with BM‐MSCs. Moreover, AT‐MSCs showed a higher level of secretion of cytokines that have been implicated in the immunomodulatory modes of action of multipotent stromal cells, such as interleukin‐6 and transforming growth factor‐β1. This is correlated with higher metabolic activity of AT‐MSCs compared with BM‐MSCs. We conclude that the immunomodulatory capacities of BM‐MSCs and AT‐MSCs are similar, but that differences in cytokine secretion cause AT‐MSCs to have more potent immunomodulatory effects than BM‐MSCs. Therefore, lower numbers of AT‐MSCs evoke the same level of immunomodulation. These data indicate that AT‐MSCs can be considered as a good alternative to BM‐MSCs for immunomodulatory therapy.
This study systematically compared the immunomodulatory capacities of adipose tissue‐derived multipotent stromal cells (AT‐MSCs) and bone marrow‐derived multipotent stromal cells (BM‐MSCs) derived from age‐matched donors. It was found that BM‐MSCs and AT‐MSCs show functionally similar immunomodulatory effects, but with a different dose‐response curve, in favor of AT‐MSCs. AT‐MSCs can be considered as a good alternative to BM‐MSCs for immunomodulatory therapy.
Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, ...and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.
Hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitors (MPPs) are routinely isolated using various markers but remain heterogeneous. Here we show that four SLAM family markers, ...CD150, CD48, CD229, and CD244, can distinguish HSCs and MPPs from restricted progenitors and subdivide them into a hierarchy of functionally distinct subpopulations with stepwise changes in cell-cycle status, self-renewal, and reconstituting potential. CD229 expression largely distinguished lymphoid-biased HSCs from rarely dividing myeloid-biased HSCs, enabling prospective enrichment of these HSC subsets. Differences in CD229 and CD244 expression resolved CD150−CD48−/lowLineage−/lowSca-1+c-Kit+ cells into a hierarchy of highly purified MPPs that retained erythroid and platelet potential but exhibited progressive changes in mitotic activity and reconstituting potential. Use of these markers, and reconstitution assays, showed that conditional deletion of Scf from endothelial cells and perivascular stromal cells eliminated the vast majority of bone marrow HSCs, including nearly all CD229−/low HSCs, demonstrating that quiescent HSCs are maintained by a perivascular niche.
•LSK cells can be subdivided into a hierarchy of 7 populations using 4 SLAM markers•CD229 expression distinguishes functionally distinct subsets of HSCs and MPPs•CD229− HSCs are mainly myeloid biased and more quiescent than CD229+ HSCs•Most HSCs, including CD229− quiescent HSCs, are maintained by a perivascular niche
Expansion of the range of SLAM family marker combinations allows prospective enrichment of distinct subpopulations of hematopoietic stem cells and multipotent progenitors.
Cyclins, cyclin-dependent kinases and other components of the core cell cycle machinery drive cell division. Growing evidence indicates that this machinery operates in a distinct fashion in some ...mammalian stem cell types, such as pluripotent embryonic stem cells. In this Review, we discuss our current knowledge of how cell cycle proteins mechanistically link cell proliferation, pluripotency and cell fate specification. We focus on embryonic stem cells, induced pluripotent stem cells and embryonic neural stem/progenitor cells.
Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular ...state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f + cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell—based therapeutics.
NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of ...peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.
EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this ...process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.
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