Transit-amplifying Progenitors Research Articles

The Protein Tyrosine Phosphatase Receptor Delta Regulates Developmental Neurogenesis

PTPRD is a receptor protein tyrosine phosphatase that is genetically associated with neurodevelopmental disorders. Here, we asked whether Ptprd mutations cause aberrant neural development by perturbing neurogenesis in the murine cortex. We show that loss of Ptprd causes increases in neurogenic transit-amplifying intermediate progenitor cells and cortical neurons and perturbations in neuronal localization. These effects are intrinsic to neural precursor cells since acute Ptprd knockdown causes similar perturbations. PTPRD mediates these effects by dephosphorylating receptor tyrosine kinases, including TrkB and PDGFRβ, and loss of Ptprd causes the hyperactivation of TrkB and PDGFRβ and their downstream MEK-ERK signaling pathway in neural precursor cells. Moreover, inhibition of aberrant TrkB or MEK activation rescues the increased neurogenesis caused by knockdown or homozygous loss of Ptprd. These results suggest that PTPRD regulates receptor tyrosine kinases to ensure appropriate numbers of intermediate progenitor cells and neurons, suggesting a mechanism for its genetic association with neurodevelopmental disorders.

Paralytic, the Drosophila voltage-gated sodium channel, regulates proliferation of neural progenitors.

Proliferating cells, typically considered "nonexcitable," nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.

The transcription factor odd-paired regulates temporal identity in transit-amplifying neural progenitors via an incoherent feed-forward loop

Neural progenitors undergo temporal patterning to generate diverse neurons in a chronological order. This process is well-studied in the developing Drosophila brain and conserved in mammals. During larval stages, intermediate neural progenitors (INPs) serially express Dichaete (D), grainyhead (Grh) and eyeless (Ey/Pax6), but how the transitions are regulated is not precisely understood. Here, we developed a method to isolate transcriptomes of INPs in their distinct temporal states to identify a complete set of temporal patterning factors. Our analysis identifies odd-paired (opa), as a key regulator of temporal patterning. Temporal patterning is initiated when the SWI/SNF complex component Osa induces D and its repressor Opa at the same time but with distinct kinetics. Then, high Opa levels repress D to allow Grh transcription and progress to the next temporal state. We propose that Osa and its target genes opa and D form an incoherent feedforward loop (FFL) and a new mechanism allowing the successive expression of temporal identities.

The A30P α-synuclein mutation decreases subventricular zone proliferation.

Parkinson's disease (PD) is associated with olfactory defects in addition to dopaminergic degeneration. Dopaminergic signalling is necessary for subventricular zone (SVZ) proliferation and olfactory bulb (OB) neurogenesis. Alpha-synuclein (α-syn or Snca) modulates dopaminergic neurotransmission, and SNCA mutations cause familial PD, but how α-syn and its mutations affect adult neurogenesis is unclear. To address this, we studied a bacterial artificial chromosome transgenic mouse expressing the A30P SNCA familial PD point mutation on an Snca-/- background. We confirmed that the SNCA-A30P transgene recapitulates endogenous α-syn expression patterns and levels by immunohistochemical detection of endogenous α-syn in a wild-type mouse and transgenic SNCA-A30P α-syn protein in the forebrain. The number of SVZ stem cells (BrdU+GFAP+) was decreased in SNCA-A30P mice, whereas proliferating (phospho-histone 3+) cells were decreased in Snca-/- and even more so in SNCA-A30P mice. Similarly, SNCA-A30P mice had fewer Mash1+ transit-amplifying SVZ progenitor cells but Snca-/- mice did not. These data suggest the A30P mutation aggravates the effect of Snca loss in the SVZ. Interestingly, calbindin+ and calretinin (CalR)+ periglomerular neurons were decreased in both Snca-/-, and SNCA-A30P mice but tyrosine hydroxylase+ periglomerular OB neurons were only decreased in Snca-/- mice. Cell death decreased in the OB granule layer of Snca-/- and SNCA-A30P mice. In the same region, CalR+ numbers increased in Snca-/- and SNCA-A30P mice. Thus, α-syn loss and human A30P SNCA decrease SVZ proliferation, cell death in the OB and differentially alter interneuron numbers. Similar disruptions in human neurogenesis may contribute to the olfactory deficits, which are observed in PD.

Rate of Progression through a Continuum of Transit-Amplifying Progenitor Cell States Regulates Blood Cell Production

The nature of cell-state transitions during the transit-amplifying phases of many developmental processes-hematopoiesis in particular-is unclear. Here, we use single-cell RNA sequencing to demonstrate a continuum of transcriptomic states in committed transit-amplifying erythropoietic progenitors, which correlates with a continuum of proliferative potentials in these cells. We show that glucocorticoids enhance erythrocyte production by slowing the rate of progression through this developmental continuum of transit-amplifying progenitors, permitting more cell divisions prior to terminal erythroid differentiation. Mechanistically, glucocorticoids prolong expression of genes that antagonize and slow induction of genes that drive terminal erythroid differentiation. Erythroid progenitor daughter cell pairs have similar transcriptomes with or without glucocorticoid stimulation, indicating largely symmetric cell division. Thus, the rate of progression along a developmental continuum dictates the absolute number of erythroid cells generated from each transit-amplifying progenitor, suggesting a paradigm for regulating the total output of differentiated cells in numerous other developmental processes.

Increasing Neural Stem Cell Division Asymmetry and Quiescence Are Predicted to Contribute to the Age-Related Decline in Neurogenesis

Adult murine neural stem cells (NSCs) generate neurons in drastically declining numbers with age. How cellular dynamics sustain neurogenesis and how alterations with age may result in this decline are unresolved issues. We therefore clonally traced NSC lineages using confetti reporters in young and middle-aged adult mice. To understand the underlying mechanisms, we derived mathematical models that explain observed clonal cell type abundances. The best models consistently show self-renewal of transit-amplifying progenitors and rapid neuroblast cell cycle exit. In middle-aged mice, we identified an increased probability of asymmetric stem cell divisions at the expense of symmetric differentiation, accompanied by an extended persistence of quiescence between activation phases. Our model explains existing longitudinal population data and identifies particular cellular properties underlying adult NSC homeostasis and the aging of this stem cell compartment.

The intestinal epithelial response to damage.

The constant renewal of the intestinal epithelium is fueled by intestinal stem cells (ISCs) lying at the base of crypts, and these ISCs continuously give rise to transit-amplifying progenitor cells during homeostasis. Upon injury and loss of ISCs, the epithelium has the ability to regenerate by the dedifferentiation of progenitor cells that then regain stemness and repopulate the pool of ISCs. Epithelial cells receive cues from immune cells, mesenchymal cells and the microbiome to maintain homeostasis. This review focuses on the response of the epithelium to damage and the interplay between the different intestinal compartments.

IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model

Background & AimsCrohn’s disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn’s disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal.MethodsA screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo.ResultsHigh IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal–associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs.ConclusionsIncreased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.

The tumor suppressor Brat controls neuronal stem cell lineages by inhibiting Deadpan and Zelda.

The TRIM-NHL protein Brain tumor (Brat) acts as a tumor suppressor in the Drosophila brain, but how it suppresses tumor formation is not completely understood. Here, we combine temperature-controlled brat RNAi with transcriptome analysis to identify the immediate Brat targets in Drosophila neuroblasts. Besides the known target Deadpan (Dpn), our experiments identify the transcription factor Zelda (Zld) as a critical target of Brat. Our data show that Zld is expressed in neuroblasts and required to allow re-expression of Dpn in transit-amplifying intermediate neural progenitors. Upon neuroblast division, Brat is enriched in one daughter cell where its NHL domain directly binds to specific motifs in the 3'UTR of dpn and zld mRNA to mediate their degradation. In brat mutants, both Dpn and Zld continue to be expressed, but inhibition of either transcription factor prevents tumorigenesis. Our genetic and biochemical data indicate that Dpn inhibition requires higher Brat levels than Zld inhibition and suggest a model where stepwise post-transcriptional inhibition of distinct factors ensures sequential generation of fates in a stem cell lineage.

Mobilizing Transit-Amplifying Cell-Derived Ectopic Progenitors Prevents Hair Loss from Chemotherapy or Radiation Therapy.

Genotoxicity-induced hair loss from chemotherapy and radiotherapy is often encountered in cancer treatment, and there is a lack of effective treatment. In growing hair follicles (HF), quiescent stem cells (SC) are maintained in the bulge region, and hair bulbs at the base contain rapidly dividing, yet genotoxicity-sensitive transit-amplifying cells (TAC) that maintain hair growth. How genotoxicity-induced HF injury is repaired remains unclear. We report here that HFs mobilize ectopic progenitors from distinct TAC compartments for regeneration in adaptation to the severity of dystrophy induced by ionizing radiation (IR). Specifically, after low-dose IR, keratin 5+ basal hair bulb progenitors, rather than bulge SCs, were quickly activated to replenish matrix cells and regenerated all concentric layers of HFs, demonstrating their plasticity. After high-dose IR, when both matrix and hair bulb cells were depleted, the surviving outer root sheath cells rapidly acquired an SC-like state and fueled HF regeneration. Their progeny then homed back to SC niche and supported new cycles of HF growth. We also revealed that IR induced HF dystrophy and hair loss and suppressed WNT signaling in a p53- and dose-dependent manner. Augmenting WNT signaling attenuated the suppressive effect of p53 and enhanced ectopic progenitor proliferation after genotoxic injury, thereby preventing both IR- and cyclophosphamide-induced alopecia. Hence, targeted activation of TAC-derived progenitor cells, rather than quiescent bulge SCs, for anagen HF repair can be a potential approach to prevent hair loss from chemotherapy and radiotherapy. Cancer Res; 77(22); 6083-96. ©2017 AACR.

Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone.

In the adult mammalian brain, neural stem cells (NSCs) generate new neurons throughout the mammal's lifetime. The balance between quiescence and active cell division among NSCs is crucial in producing appropriate numbers of neurons while maintaining the stem cell pool for a long period. The Notch signaling pathway plays a central role in both maintaining quiescent NSCs (qNSCs) and promoting cell division of active NSCs (aNSCs), although no one knows how this pathway regulates these apparently opposite functions. Notch1 has been shown to promote proliferation of aNSCs without affecting qNSCs in the adult mouse subependymal zone (SEZ). In this study, we found that Notch3 is expressed to a higher extent in qNSCs than in aNSCs while Notch1 is preferentially expressed in aNSCs and transit-amplifying progenitors in the adult mouse SEZ. Furthermore, Notch3 is selectively expressed in the lateral and ventral walls of the SEZ. Knockdown of Notch3 in the lateral wall of the adult SEZ increased the division of NSCs. Moreover, deletion of the Notch3 gene resulted in significant reduction of qNSCs specifically in the lateral and ventral walls, compared with the medial and dorsal walls, of the lateral ventricles. Notch3 deletion also reduced the number of qNSCs activated after antimitotic cytosine β-D-arabinofuranoside (Ara-C) treatment. Importantly, Notch3 deletion preferentially reduced specific subtypes of newborn neurons in the olfactory bulb derived from the lateral walls of the SEZ. These results indicate that Notch isoforms differentially control the quiescent and proliferative steps of adult SEZ NSCs in a domain-specific manner.SIGNIFICANCE STATEMENT In the adult mammalian brain, the subependymal zone (SEZ) of the lateral ventricles is the largest neurogenic niche, where neural stem cells (NSCs) generate neurons. In this study, we found that Notch3 plays an important role in the maintenance of quiescent NSCs (qNSCs), while Notch1 has been reported to act as a regulator of actively cycling NSCs. Furthermore, we found that Notch3 is specifically expressed in qNSCs located in the lateral and ventral walls of the lateral ventricles and regulates neuronal production of NSCs in a region-specific manner. Our results indicate that Notch3, by maintaining the quiescence of a subpopulation of NSCs, confers a region-specific heterogeneity among NSCs in the adult SEZ.

Quantitative microscopy of the Drosophila ovary shows multiple niche signals specify progenitor cell fate

Adult stem cells commonly give rise to transit-amplifying progenitors, whose progeny differentiate into distinct cell types. It is unclear if stem cell niche signals coordinate fate decisions within the progenitor pool. Here we use quantitative analysis of Wnt, Hh, and Notch signalling reporters and the cell fate markers Eyes Absent (Eya) and Castor (Cas) to study the effects of hyper-activation and loss of niche signals on progenitor development in the Drosophila ovary. Follicle stem cell (FSC) progeny adopt distinct polar, stalk, and main body cell fates. We show that Wnt signalling transiently inhibits expression of the main body cell fate determinant Eya, and Wnt hyperactivity strongly biases cells towards polar and stalk fates. Hh signalling independently controls the proliferation to differentiation transition. Notch is permissive but not instructive for differentiation of multiple cell types. These findings reveal that multiple niche signals coordinate cell fates and differentiation of progenitor cells.

Seven in Absentia E3 Ubiquitin Ligases: Central Regulators of Neural Cell Fate and Neuronal Polarity.

During neural development, neural precursors transition from a proliferative state within their germinal niches to a migratory state as they relocate to their final laminar positions. Transitions across these states are coupled with dynamic alterations in cellular polarity. This key feature can be seen throughout the developing vertebrate brain, in which neural stem cells give rise to multipolar or unpolarized transit-amplifying progenitors. These transit-amplifying progenitors then expand to give rise to mature neuronal lineages that become polarized as they initiate radial migration to their final laminar positions. The conventional understanding of the cellular polarity regulatory program has revolved around signaling cascades and transcriptional networks. In this review, we discuss recent discoveries concerning the role of the Siah2 ubiquitin ligase in initiating neuronal polarity during cerebellar development. Given the unique features of Siah ubiquitin ligases, we highlight some of the key substrates that play important roles in cellular polarity and propose a function for the Siah ubiquitin proteasome pathway in mediating a post-translational regulatory network to control the onset of polarization.

Radial glia in the ventral telencephalon.

The ventral telencephalon is the developmental origin of the basal ganglia and the source of neuronal and glial cells that integrate into developing circuits in other areas of the brain. Radial glia in the embryonic subpallium give rise to an enormous diversity of mature cell types, either directly or through other transit-amplifying progenitors. Here, we review current knowledge about these subpallial neural stem cells and their progeny, focusing on the period of neurogenesis. We describe their cell biological features and the extrinsic and intrinsic molecular codes that guide their fate specification in defined temporal and spatial sequences. We also discuss the role of clonal lineage in the organization and specification of mature neurons.

Cell-autonomous requirement for mammalian target of rapamycin (Mtor) in spermatogonial proliferation and differentiation in the mouse†.

Spermatogonial stem cells must balance self-renewal with production of transit-amplifying progenitors that differentiate in response to retinoic acid (RA) before entering meiosis. This self-renewal vs. differentiation fate decision is critical for maintaining tissue homeostasis, as imbalances cause defects that can lead to human testicular cancer or infertility. Little is currently known about the program of differentiation initiated by RA, and the pathways and proteins involved are poorly defined. We recently found that RA stimulation of the Phosphatidylinositol 3-kinase (PI3K)/AKT/Mammalian target of rapamycin (mTOR) kinase signaling pathway is required for differentiation, and that short-term inhibition of mTOR complex 1 (mTORC1) by rapamycin blocked spermatogonial differentiation in vivo and prevented RA-induced translational activation. Since this phenotype resulted from global inhibition of mTORC1, we created conditional germ cell knockout mice to investigate the germ cell-autonomous role of MTOR in spermatogonial differentiation. MTOR germ cell KO mice were viable and healthy, but testes from neonatal (postnatal day (P)8), juvenile (P18), and adult (P > 60) KO mice were smaller than littermate controls, and no sperm were produced in adult testes. Histological and immunostaining analyses revealed that spermatogonial differentiation was blocked, and no spermatocytes were formed at any of the ages examined. Although spermatogonial proliferation was reduced in the neonatal testis, it was blocked altogether in the juvenile and adult testis. Importantly, a small population of self-renewing undifferentiated spermatogonia remained in adult testes. Taken together, these results reveal that MTOR is dispensable for the maintenance of undifferentiated spermatogonia, but is cell autonomously required for their proliferation and differentiation.

The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in theSCO histological phenotype. We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. N/A. There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests.

REST regulation of gene networks in adult neural stem cells.

Adult hippocampal neural stem cells generate newborn neurons throughout life due to their ability to self-renew and exist as quiescent neural progenitors (QNPs) before differentiating into transit-amplifying progenitors (TAPs) and newborn neurons. The mechanisms that control adult neural stem cell self-renewal are still largely unknown. Conditional knockout of REST (repressor element 1-silencing transcription factor) results in precocious activation of QNPs and reduced neurogenesis over time. To gain insight into the molecular mechanisms by which REST regulates adult neural stem cells, we perform chromatin immunoprecipitation sequencing and RNA-sequencing to identify direct REST target genes. We find REST regulates both QNPs and TAPs, and importantly, ribosome biogenesis, cell cycle and neuronal genes in the process. Furthermore, overexpression of individual REST target ribosome biogenesis or cell cycle genes is sufficient to induce activation of QNPs. Our data define novel REST targets to maintain the quiescent neural stem cell state.

Stem and progenitor cells of the mammalian olfactory epithelium: Taking poietic license.

The capacity of the olfactory epithelium (OE) for lifelong neurogenesis and regeneration depends on the persistence of neurocompetent stem cells, which self-renew as well as generating all of the cell types found within the nasal epithelium. This Review focuses on the types of stem and progenitor cells in the epithelium and their regulation. Both horizontal basal cells (HBCs) and some among the population of globose basal cells (GBCs) are stem cells, but the two types plays vastly different roles. The GBC population includes the basal cells that proliferate in the uninjured OE and is heterogeneous with respect to transcription factor expression. From upstream in the hierarchy to downstream, GBCs encompass 1) Sox2+ /Pax6+ stem-like cells that are totipotent and self-renew over the long term, 2) Ascl1+ transit-amplifying progenitors with a limited capacity for expansive proliferation, and 3) Neurog1+ /NeuroD1+ immediate precursor cells that make neurons directly. In contrast, the normally quiescent HBCs are activated to multipotency and proliferate when sustentacular cells are killed, but not when only OSNs die, indicating that HBCs are reserve stem cells that respond to severe epithelial injury. The master regulator of HBC activation is the ΔN isoform of the transcription factor p63; eliminating ΔNp63 unleashes HBC multipotency. Notch signaling, via Jagged1 ligand on Sus cells and Notch1 and Notch2 receptors on HBCs, is likely to play a major role in setting the level of p63 expression. Thus, ΔNp63 becomes a potential therapeutic target for reversing the neurogenic exhaustion characteristic of the aged OE. J. Comp. Neurol. 525:1034-1054, 2017. © 2016 Wiley Periodicals, Inc.

3D Culture Supports Long-Term Expansion of Mouse and Human Nephrogenic Progenitors

Transit-amplifying nephron progenitor cells (NPCs) generate all of the nephrons of the mammalian kidney during development. Their limited numbers, poor invitro expansion, and difficult accessibility in humans have slowed basic and translational research into renal development and diseases. Here, we show that with appropriate 3D culture conditions, it is possible to support long-term expansion of primary mouse and human fetal NPCs as well as NPCs derived from human induced pluripotent stem cells (iPSCs). Expanded NPCs maintain genomic stability, molecular homogeneity, and nephrogenic potential invitro, exvivo, and invivo. Cultured NPCs are amenable to gene targeting and can form nephron organoids that engraft invivo, functionally couple to the host's circulatory system, and produce urine-like metabolites via filtration. Together, these findings provide a technological platform for studying human nephrogenesis, modeling and diagnosing renal diseases, and drug discovery.

Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

SummaryHuman skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations.