In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing in?vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in?vivo properties of adult neural stem cells.     

Proteomic cornerstones of hematopoietic stem cell differentiation: distinct signatures of multipotent progenitors and myeloid committed cells

Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage commitment.     

Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine

Tight regulation of self-renewal and differentiation of adult stem cells ensures that tissues are properly maintained. In the Drosophila intestine, both commitment, i.e. exit from self-renewal, and terminal differentiation are controlled by Notch signaling. Here, we show that distinct requirements for Notch activity exist: commitment requires high Notch activity, whereas terminal differentiation can occur with lower Notch activity. We identified the gene GDP-mannose 4,6-dehydratase (Gmd), a modulator of Notch signaling, as being required for commitment but dispensable for terminal differentiation. Gmd loss resulted in aberrant, self-renewing stem cell divisions that generated extra ISC-like cells defective in Notch reporter activation, as well as wild-type-like cell divisions that produced properly terminally differentiated cells. Lowering Notch signaling using additional genetic means, we provided further evidence that commitment has a higher Notch signaling requirement than terminal differentiation. Our work suggests that a commitment requirement for high-level Notch activity safeguards the stem cells from loss through differentiation, revealing a novel role for the importance of Notch signaling levels in this system.     

Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells

The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.     

Notch promotes neural lineage entry by pluripotent embryonic stem cells

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.     

Temporal transcriptional profiling of somatic and germ cells reveals biased lineage priming of sexual fate in the fetal mouse gonad

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.     

The TBC1D15 Oncoprotein Controls Stem Cell Self-Renewal through Destabilization of the Numb-p53 Complex

Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKCζ, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.     

Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment

We demonstrate here that "promiscuous" expression of myeloid or lymphoid genes precedes lineage commitment in hematopoiesis. Prospectively purified single common myeloid progenitors (CMPs) coexpress myelo-erythroid but not lymphoid genes, whereas single common lymphoid progenitors (CLPs) coexpress T and B lymphoid but not myeloid genes. Genes unrelated to the adopted lineage are downregulated in bipotent and monopotent descendants of CMPs and CLPs. Promiscuous gene expression does not alter the biological potential of multipotent progenitors: CMPs with an activated endogenous M lysozyme locus yield normal proportions of myelo-erythroid colonies, and CLPs expressing the pre-T cell receptor alpha gene differentiate into normal numbers of B cells. Thus, the accessibility for multiple myeloid or lymphoid programs promiscuously may allow flexibility in fate commitments at these multipotent stages.     

Isolation of neural crest-derived stem cells from rat embryonic mandibular processes

BACKGROUND INFORMATION: Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. RESULTS: LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. CONCLUSIONS: These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.     

Chronic myeloid leukemia: proving ground for cancer stem cells

A central question in cancer biology is whether malignancy arises in self-renewing tissue stem cells that suffer oncogene activation or in differentiated cells that acquire properties of unremitting self-renewal? In two papers, Weissman and colleagues document both mechanisms: chronic leukemia arising by mutation affecting the hematopoietic stem cell (HSC) and acute leukemia evolving from committed granulocyte-macrophage progenitors that have acquired the self-renewal machinery of HSCs.