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Prospective isolation of defined cell types is critical for the functional study of stem cells, especially in primary human tissues. Here, we present a protocol for purifying 10 transcriptomically and functionally distinct neural stem and progenitor cell types from the developing human brain using fluorescence-activated cell sorting. We describe steps for tissue dissociation, staining, and cell sorting as well as downstream functional experiments for measuring clonogenicity, differentiation, and engraftment potential of purified populations. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023). 1 ., Competing Interests: Declaration of interests D.D.L., J.Q.H., R.S., N.U., and I.L.W. are listed as inventors on a pending patent related to this protocol. I.L.W. is a co-founder of Bitterroot Bio, Inc. and Pheast, Inc., neither of which are related to the current protocol. I.L.W. was an initial co-founder and N.U. a former employee of StemCells, Inc., but they are not currently consultants or employees at the company or its successor., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mechanisms that sustain stem cells are fundamental to tissue maintenance. Here, we identify "cell islands" (CIs) as a niche for putative germ and somatic stem cells in Botryllus schlosseri, a colonial chordate that undergoes weekly cycles of death and regeneration. Cells within CIs express markers associated with germ and somatic stem cells and gene products that implicate CIs as signaling centers for stem cells. Transplantation of CIs induced long-term germline and somatic chimerism, demonstrating self-renewal and pluripotency of CI cells. Cell labeling and in vivo time-lapse imaging of CI cells reveal waves of migrations from degrading CIs into developing buds, contributing to soma and germline development. Knockdown of cadherin, which is highly expressed within CIs, elicited the migration of CI cells to circulation. Piwi knockdown resulted in regeneration arrest. We suggest that repeated trafficking of stem cells allows them to escape constraints imposed by the niche, enabling self-preservation throughout life., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A report by the International Society for Stem Cell Research (ISSCR)'s Task Force on Unproven Stem Cell Treatments outlines development of resources for patients, their families, and physicians seeking information on stem cell treatments., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1(-/)(-) mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology. Grafted mice displayed reduced autofluorescent lipofuscin, significant neuroprotection of host hippocampal and cortical neurons, and delayed loss of motor coordination. Early intervention with cellular transplants of hCNS-SCns into the brains of INCL patients may supply a continuous and long-lasting source of the missing PPT1 and provide some therapeutic benefit through protection of endogenous neurons. These data provide the experimental basis for human clinical trials with these banked hCNS-SCns.

The aging of tissue-specific stem cell and progenitor cell compartments is believed to be central to the decline of tissue and organ integrity and function in the elderly. Here, we examine evidence linking stem cell dysfunction to the pathophysiological conditions accompanying aging, focusing on the mechanisms underlying stem cell decline and their contribution to disease pathogenesis.

Stem cells are highly conserved biological units of development and regeneration. Here we formally demonstrate that stem cell lineages are also legitimate units of natural selection. In a colonial ascidian, Botryllus schlosseri, vascular fusion between genetically distinct individuals results in cellular parasitism of somatic tissues, gametes, or both. We show that genetic hierarchies of somatic and gametic parasitism following fusion can be replicated by transplanting cells between colonies. We prospectively isolate a population of multipotent, self-renewing stem cells that retain their competitive phenotype upon transplantation. Their single-cell contribution to either somatic or germline fates, but not to both, is consistent with separate lineages of somatic and germline stem cells or pluripotent stem cells that differentiate according to the niche in which they land. Since fusion is restricted to individuals that share a fusion/histocompatibility allele, these data suggest that histocompatibility genes in Botryllus evolved to protect the body from parasitic stem cells usurping asexual or sexual inheritance.

Recent years have seen much excitement over the possibility that adult mammalian stem cells may be capable of differentiating across tissue lineage boundaries, and as such may represent novel, accessible, and very versatile effectors of therapeutic tissue regeneration. Yet studies proposing such "plasticity" of adult somatic stem cells remain controversial, and in general, existing evidence suggests that in vivo such unexpected transformations are exceedingly rare and in some cases can be accounted for by equally unexpected alternative explanations.

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schiosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Bottyllus' endostyle region as an adult somatic stem cell niche. [ABSTRACT FROM AUTHOR]

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential. [ABSTRACT FROM AUTHOR]

Discusses the history and advances in stem cells research. Examination of hematopoietic stem cells; Regeneration of the hematolymphoid system; Progeny of cell divisions; Development processes associated with stem and progenitor cells.

Self-renewal pathways crucial for maintaining stem cells are deregulated in cancer, raising the spectre that cancer therapies targeting such pathways might also ablate normal stem cells. As report in a recent Nature paper, this may not be the case for the tumor suppressor protein Pten, which drives the self-renewal of normal hematopoietic stem cells and the formation of leukemia cells through different mechanisms. [Copyright &y& Elsevier]

Summary: A model of hematopoietic development wherein multipotentiality is conserved until segregation of myeloid and lymphoid potential has recently been challenged, proposing that megakaryocyte/erythrocyte (MegE) potential is lost in Flk2/Flt3-expressing early progenitors. Here, we used sensitive in vivo approaches to quantitatively and kinetically assess the MegE potential of hematopoietic stem cells and various Flk2+ early progenitors and compared it with the MegE potential of downstream committed myeloid and lymphoid progenitors and with their ability to give rise to mature myelomonocytic and lymphoid cells. We demonstrate that Flk2+ early progenitors retain MegE potential in vivo both at the population and clonal levels. These results indicate that Flk2 expression by early progenitors is not at the expense of full multipotency and support the current model of hematopoietic development with segregation of myeloid and lymphoid lineages from multipotent progenitors. [Copyright &y& Elsevier]