Genetic studies have identified a number of stemcell-intrinsic factors that are important for regulating stemcell-niche adhesion. Notably, many of these factors act by modulating E-cadherin expression or function in stem cells (Fig. 4A), although some function by regulating integrin activity (Fig. 4B). In the Drosophila ovary, Lissencephaly-1 (Lis-1), which is encoded by the Drosophila homolog of the causative gene (LIS1, also known as PAFAH1B1) for the human disease lissencephaly, is required in GSCs to maintain E-cadherin accumulation at the GSC-niche junction via an unknown mechanism (Chen et al., 2010). In addition, Lis-1 is required for centrosome positioning in Drosophila ovarian and testicular GSCs as well as for spindle orientation in neuroblasts and mouse neural progenitor cells (Chen et al., 2010; Siller and Doe, 2008; Sitaram et al., 2012; Yingling et al., 2008). However, it remains unclear whether centrosome positioning and spindle orientation are connected with the adhesion role of Lis-1. Eukaryotic initiation factor 4A (eIF-4A) is also required for maintaining E-cadherin expression in GSCs. By contrast, the differentiation factors Bam and Bgcn can repress E- cadherin translation, probably through the E-cadherin 3 ⬘ UTR (Jin et al., 2008; Shen et al., 2009), and eIF-4A functions through direct interaction with Bam, antagonizing Bam-mediated translational repression of E-cadherin (Shen et al., 2009). Additionally, Poly(ADP-ribose) glycohydrolase (Parg) and the heterogeneous nuclear ribonucleoprotein Hrp38 (also known as Hrb98DE) are required for GSC maintenance and act by sustaining E-cadherin expression through translational regulation (Ji and Tulin, 2012); Parg degrades poly(ADP-ribose), which binds to Hrp38 to prevent Hrp38 association with the 5 ⬘ UTR of E-cadherin. In FSCs, Cyclin E is required for niche anchorage and it is proposed to act by maintaining E-cadherin expression; Cyclin E mutant FSCs are lost from the niche, but the forced expression of E-cadherin can restore niche retention, suggesting that the FSC cell cycle is coupled with niche adhesion (Wang and Kalderon, 2009). Interestingly, the FSC loss phenotype caused by many other mutations, such as SCAR, smoothened, Coprox or Actin-related protein 2/3 complex subunit 1 (ArpC1), can be partially rescued by forced expression of E- cadherin, indicating that E-cadherin-mediated cell adhesion between FSCs and their niche might represent the focal point for FSC regulation (Wang et al., 2012).
Strategies to engineer a stemcellniche. There are a variety of approaches to engineer and control individual niche components. These strategies can be multiplexed to produce hybrid devices that simultaneously provide macroscopic (e.g., O 2 -controlled bioreactors)
C-terminally tagged Zfh1 fusion proteins are functional Visualisation of expression patterns by GFP reporters, and temporally and spatially controlled expression using the Gal4 system, are key elements of the Drosophila toolbox. The zfh1 locus possesses two transcription starts, separated by about 17 kb. The respective transcripts are spliced to common 3 ′ exons (Fig. 1D), giving rise to two isoforms that are both expressed in the testis (Fig. S1). The first, zfh1-RA, encodes a 747 amino acid protein containing seven C2H2 zinc fingers and a central homeodomain, whereas the 1045 amino acid protein, encoded by zfh1-RB, contains two additional N-terminal zinc fingers and a polyQ region. We therefore targeted GFP to the common C terminus of both isoforms (Fig. 1D) by Crispr/Cas9-mediated recombination (Gratz et al., 2014; Port et al., 2014). The resulting zfh1::GFP knock-in lines were homozygous, viable and fertile. Zfh1::GFP marks the nuclei of somatic cells in the vicinity of the hub (32.7±4.2 GFP + cells/testis, Fig. 1. Endogenous Zfh1 expression and Crispr/Cas9-generated reagents. (A) Schematic of the stemcellniche of the Drosophila testis tip. Zfh1 (magenta) marks the nuclei of the CySCs and, at lower levels, their immediate progeny. (B) CySCs are recognised by their Zfh1 + nuclei (blue) and their position adjacent to
The use of organoid culture has helped to identify important niche components and has led to a deeper understanding of how the niche controls stemcell activity. The ability to manipulate organoid formation in vitro has enabled careful dissection of niche requirements, as demonstrated by reaggregation experiments that identified Paneth cells as a key member of the intestinal stemcellniche (Sato et al., 2011). Such experiments in other tissues will be useful for identifying the cell types that comprise the ʻ minimal niche ’ , that is, the components that are necessary and sufficient for stemcell maintenance and tissue self-renewal. In addition to identifying feedback pathways from daughter to parent cells, organoids can also help to identify feedforward pathways between parent and daughter cells that help direct and maintain differentiation, as discussed in the case of Notch signaling in airway basal stem cells (Pardo-Saganta et al., 2015; Rock et al., 2011). Controlled manipulation of organoid composition, including the numbers of stem cells and specific numbers and types of differentiated daughter cells, will allow interrogation of the feedforward and feedback loops that control cell fate in other tissues. Emerging methods in organoid engineering will accelerate progress in our understanding of the stemcellniche by providing powerful means of interrogating organoids at the single cell level, as well as a means to guide the morphogenesis of organoids with significantly greater precision. This is crucial if we are to be able to use organoids in any meaningful way to address fundamental questions in developmental and regenerative biology (Huch et al., 2017). These emerging technologies will facilitate studies that aim to address key concepts in stemcell biology, such as the influence of the niche on cell plasticity, as well as how the physical properties of the stemcellniche direct stemcell self-organization and, in turn, stemcell fate. For example, progenitors that are restricted to the enterocyte lineage during normal intestinal homeostasis can
The traditional paradigm of a unidirectional, hierarchal differen- tiation trajectory – beginning with a multipotent self-renewing stemcell and proceeding through transit-amplifying cell stages before transitioning into the terminal differentiated state – was uncovered in the embryonic and hematopoietic stemcell fields (Weissman, 2000). These concepts have further been applied to epithelial tissue-resident stem cells. However, accumulating evidence suggests that stemness, especially in the context of epithelial tissues, is a bidirectional, dynamic state that is largely governed by the stemcellniche, allowing plasticity and adaptability to changing conditions (Chacón-Martínez et al., 2017; Ritsma et al., 2014; Rompolas et al., 2013; Sun et al., 2014; Takeda et al., 2011). These discoveries highlight the importance of the biochemical composition and biophysical architecture of the niche, which influence stemcell state and fate (Morrison and Spradling, 2008; Scadden, 2014). Given this central role of the niche in regulating epithelial stemcell behavior, the identification of niche factors and signaling pathways that can promote stemcell function or enrich for cells with stemcell properties could have significant implications in regenerative medicine and tissue engineering. In this Review, we discuss how niche inputs regulate stemcell functions during morphogenesis, homeostasis and regeneration, focusing mainly on three well-studied mammalian systems: the lung, gastrointestinal tract and skin epithelia. We first provide an overview of the stemcell dynamics within these tissues. We then describe recent research that has identified key biochemical and biomechanical signals within the niche that control stemcell fate, and highlight how these signals facilitate stemcell heterogeneity and plasticity to ensure robust tissue function and repair.
with the hematopoietic stemcellniche in the trabecular region of the metaphysis of long bones. Shiozawa et al. elegantly demonstrated that prostate cancer cells and HSCs locate within the same niche [20,21] using a CXCR4 antagonist (AMD3100) to mobilize the HSCs, but it is not known whether this is also the case for breast cancer cells. We therefore used a similar approach to investigate whether mobilisation of HSCs from the bone niche into the circulation would increase the number of sites available for tumour cells to colonise, as outlined in Fig. 7A. To allow direct comparison with the Shiozawa study, the same dose of AMD3100 (5 mg/kg i.p) shown in their study to mobilise HSCs  was injected daily for 5 days in 12-week old mice. As shown in Fig. 7B, after 11 days of culture ex vivo there was a signiicant increase in colony numbers generated from HSC/and other progenitor cells (PCs) isolated from blood samples of AMD3100-treated mice compared to control, demonstrating successful mobilization of HSCs/PCs. Next, Vybrant-CM-DiI labelled MDA-MB-231-GFP-IV cells were injected into the tail vein of animals that had received 5 days of AMD3100 or control treatment. Animals were culled 5 days following tumour cell injection and the number and location of tumour cells in the bone marrow was compared between control and AMD3100 treated animals using two- photon microscopy.
Two LIM-homeodomain proteins of the LMX subgroup are encoded in the Drosophila genome: Lmx1a/CG32105 and Lmx1b/CG4328. Lmx1a is expressed in the LL3 eye imaginal disc and its overexpression in this tissue causes eye defects (Roignant et al., 2010; Wang et al., 2016). However, the molecular, cell biological and developmental functions of this transcription factor have yet to be described. Here, we show that Lmx1a is expressed in somatic lineages in the developing ovary. We found that its expression becomes restricted to TFs and cap cells by LL3/P0 (freshly pupariated) and is maintained in the adult stemcellniche. Analyzing a CRISPR-generated Lmx1a knockout allele as well as cell type-specific and stage-specific knockdown of Lmx1a, we have determined that Lmx1a is required for ovary development specifically in the TF-cap cellniche at the time at which it forms. We performed transcriptional profiling of developing ovaries and placed Lmx1a downstream of Bab1/2 in the specification of TF cells. We also show that without Lmx1a, several components of signaling pathways crucial to the forming niche are not properly expressed, including Hh, the transcription factors Sox100B, Engrailed and Invected, and confirmed that these genes are required in the Lmx1a lineage. Strikingly, expression of a chicken ortholog of Lmx1a in forming TF-cap cells is sufficient to rescue the Lmx1a null phenotype. We anticipate that these results will further elucidate the genetic and cell biological mechanisms underlying the establishment of the Drosophila ovary stemcellniche and provide insight into the role of LIM-HD factors in tissue development and patterning, homeostasis and disease.
Adult male Drosophila contain a pair of testes; each is a long blind-ended tube that is coiled around a seminal vesicle. The stemcellniche is located at the blind apical end of the testis. Here, GSCs divide asymmetrically to generate one cell that remains a stemcell and another, a gonialblast, that is displaced away from the niche and differentiates (Fig. 1). Each gonialblast is enveloped by two somatic cyst cells, which arise from cyst stem cells (CySCs) that also divide asymmetrically to self-renew and produce differentiating cyst cell daughters. A gonialblast progresses through four rounds of transit-amplifying divisions to produce a cluster of 16 spermatogonial cells; cytokinesis is incomplete in each division and the 16 cells remain connected by stable intercellular bridges called ring canals. These 16 spermatogonial cells progress through premeiotic S phase and then switch to a spermatocyte program of growth and gene expression; most of the gene products that are needed for the development of spermatocytes and spermatids are transcribed at this time (White-Cooper, 2010). GSCs, gonialblasts and spermatogonia are almost identical morphologically, but spermatocytes and spermatids undergo dramatic changes in both size and shape. The two cyst cells that envelop the gonialblast do not divide, but they continue to grow and encase the gonialblast and its progeny throughout spermatogenesis. At the end of spermatogenesis, the spermatids lose their interconnections and become surrounded by individual plasma membranes. Mature sperm are then released from the open end of the testis into the seminal vesicle, where they are stored until needed. Thus, the testis contains a gradient of developmental stages, from stem cells in the niche at the apical end to mature sperm at the basal end.
Why do the majority of Drosophila GSCs undergo asymmetric division if symmetric renewal plus symmetric differentiation produces the same output? As GSCs and CySCs function together within the niche during spermatogenesis, robust division orientation of both populations may enable differentiating germline cells to be generated at a rate that matches cyst cell production. Asymmetric divisions may also prevent clonal expansion of stem cells harboring harmful mutations within the niche, which can compete for niche occupancy (Issigonis et al., 2009; Johnston, 2009). However, clonal expansion may not always be harmful; mammalian niches regularly progress towards mono-clonality with stem cells exhibiting neutral drift dynamics (Klein et al., 2010; Lopez-Garcia et al., 2010; Snippert et al., 2010). Perhaps symmetrically renewing divisions are not detrimental to mammalian systems because mammalian niches are not as constrained spatially, and mammalian stem cells are often motile (Morrison and Spradling, 2008; Nakagawa et al., 2010; Yoshida et al., 2007). So far, asymmetric division in Drosophila testes correlates with optimal GSC function, as it becomes less robust with aging (Cheng et al., 2008). Whether symmetric divisions increase during aging has not been examined, but it might occur because GSCs are thought to be lost more frequently due to decreased maintenance cues (Boyle et al., 2007; Wallenfang, 2007). Interestingly, depleting STAT92E from GSCs displaces them from the hub, yet they are not lost from the tissue. Instead, they associate with BMP- producing CySCs, which probably promote GSC renewal. However, GSC division orientation is now randomized (Leatherman and DiNardo, 2010); suggesting that their output is composed of symmetric renewals and symmetric differentiation. Furthermore, APC2 mutants that affect centrosome position and E-cadherin mutants that have misoriented divisions still have wild-type GSC numbers (Inaba et al., 2010). Together, these observations suggest that the Drosophila testis stemcellniche does not require invariant asymmetric GSC division outcomes.
Several investigators have shown similarities between Sertoli cells and MSC , although MSC did not ori- ginate from Sertoli cells, at least not in in vitro experi- ments . Therefore, MSC might support or attract stemcellniche components and/or mimic the paracrine signals of absent niche cells. Accordingly, insulin-like growth factor (IGF) was present in MSC secretome and promoted testosterone production by Leydig cells in other studies [38, 42]. This hypothesis is consistent with an advanced conception of the regenerative potential of MSC , for which consideration as effectors has been replaced with consideration as regulators that transiently provide paracrine stimuli for target cells and trigger regenerative processes in tissues after damage. These roles of MSC in stemcell niches should be further investigated.
Our data are also supported by previous studies. Consti- tutive activation of the PTH receptor in osteoblasts in- creased the HSC number and activity . Ballen et al.  performed a phase I study for PTH and found that 47 % of patients with hematologic malignancies acquired ad- equate CD34 + cells with the help of PTH. Moreover, RANKL-stimulated bone-resorbing osteoclasts reduce the stemcellniche components SDF-1, SCF, and OPN in the endosteum and finally trigger HSPC mobilization, so RANKL may be used together with other mobilization agents in an extensive range of clinical HSPC transplant- ation protocols . In the present study, the CRA showed that recipient mice transplanted with circulation HSPCs from the P + R and P + R + G groups had more robust myeloid and lymphatic cell engraftment than either the CTL or G group. These findings suggest that stimula- tion of the endosteal bone marrow niche can lead to in- creased engraftment of the HSC compartment through increased expansion of the stemcell pool.
In summary, several cell types cooperate to produce secreted and membrane-bound signaling molecules controlling HSC maintenance, fate and function, thus contributing to the formation of the complex HSC-niche unit. These signal/receptor pairs include: SCF/KIT; CXCL12/CXCR4, TGF-ß/TGFß RII, Ang-1/Tie2 and thrombopoietin/MPL and several others with more fine tuning effects on HSCs [77,89-93]. The last three have been suggested to promote dormancy or hibernation, a typical feature of the most potent HSCs during homeostasis [81,94]. Future research will need to decipher the three-dimensional network of the HSC- niche unit, and to dissect the various extracellular signals and how these are translated into HSC fate and function. In addition, it will be important to unravel the architectural, cellular and molecular changes within the HSC-niche units in response to various stress situations, including bacterial and viral infections as well as chemotherapy-induced toxicity. Not only will a better understanding of these processes in mice and humans allow us to understand more clearly the many different facets of HSC biology during homeostasis and stress, but it may also provide direct clinical applications for many disease areas as well as for regenerative medicine.
Th e availability of a number of speciﬁ c cell surface markers allowed the isolation, puriﬁ cation, and func tional characterization of HSCs in vitro and in vivo, leading to the recent identiﬁ cation of a population of highly quiescent, injury-responsive, dormant HSCs. As the full nature of the HSC niche remains elusive, the challenge now is to understand whether dormant and homeostatic HSCs segregate in speciﬁ c niches or coexist in sub- sections of the same niche. Th e comparison of the hematopoietic system with other tissues characterized by high turnover, such as the epidermis and the intestinal epithelium, leads to an emerging pattern of allocation of duties between somatic stemcell subpopulations, with some of them being responsible for day-to-day mainte- nance and others being set aside for prompt repair of injury, and a similar pattern is emerging even for organs characterized by very slow turnover, such as the brain. While histological analysis and whole-mount prepara- tions provide excellent tools for performing detailed label retention and lineage-tracing analysis in the epidermis and the intestine [42,45], it is likely that further develop- ments in three-dimensional live imaging technology will be needed in order to generate a clear picture of the localization and behavior of dormant and homeostatic somatic stem cells . Th e combination of the diﬀ erent experimental approaches currently used for each tissue will likely solve the debate on the prescence of dormant stem cells and their niches.
A recent report addressed the establishment of ISC clones in the developing midgut. During Drosophila develop- ment, adult midgut progenitors (AMPs) originate from the embryonic endoderm, disperse, and divide to expand the midgut during the larval stage . Surrounding visceral muscle cells secrete ligands to activate epidermal growth factor signaling in AMPs, causing them to divide to fill the developing tissue. This close relationship between the gut epithelium and its surrounding muscle niche continues into adulthood, during which the surrounding muscle serves as the source of Wg (wingless) and Upd (unpaired) ligands in order to activate Wnt and JAK/STAT (janus kinase/signal transducer and activator of transcription) signaling in ISCs, respectively [9,20]. The dependence of ISCs on signals emanating from the surrounding muscle cells explains in part why ISCs are located basally in the epithelium.
proliferation varies among each population. The cell cycle length of slowly dividing stem cells is more than 6 days, while transit amplifying progenitor cells and neuroblasts progress through the stages of the cell cycle every 24 hours (Doetsch et al., 1999b). It was originally suggested that ependymal cells divide in vivo and function as stem cells (Johansson et al., 1999), but subsequent studies have failed to identify dividing ependymal cells in the adult SVZ (Capela and Temple, 2002; Chiasson et al., 1999; Doetsch et al., 2002; Laywell et al., 2000; Spassky et al., 2005; Carlen et al., 2009). Whether or not any ependymal cells re-enter the cell cycle at various stages of development remains highly controversial.
The Drosophila male germline system is an excellent model to study niche-stemcell biology. Extensive work in the field has demonstrated that signals provided by the niche play a crucial role in regulating the behavior of GSCs (44-46, 50-53). Among those identified signals, the JAK-STAT pathway has received most attention (44, 45). A model which involves a single niche has also been dominant in the field. The studies presented here as well as recent work in our lab emphasize the importance of another signaling pathway, BMP, and also modify the niche-stemcell model (40). We characterize the role of Magu as a novel BMP modulator specifically required for GSC maintenance. The function of Magu may also depend on its interaction with heparan sulfate proteoglycans. Previous work in the lab identified the transcription factor Zfh1 as a key regulator for both CySCs and GSCs (42). We have attempted to identify downstream targets of Zfh1 using two genome-wide approaches. Preliminary results suggest that Zfh1 can function as either a repressor or activator to control the expression of target genes. Together, these findings further reveal the complexity of the seemingly simple niche system in the fruit fly testis. Below I will discuss two interesting questions related to the Magu project. I will also address remaining questions and future experiments for the ongoing project on Zfh1.
For immunofluorescence, tibiae were collected and fixed for 2 hours in 4% paraformaldehyde. Bone decalcification was performed for 48 hours in Osteosoft (Merck Millipore), and bones were embed- ded in optical cutting temperature compound (OCT; Tissue-Tek). Endothelial cell staining in decalcified bones was performed using an APC-conjugated anti-CD31/PECAM-1 (clone 390; eBioscience) antibody and Alexa Fluor 694–conjugated anti-CD144/VE-cadherin (clone BV13; BioLegend), as described previously (7). In other experi- ments endothelial cell staining in bones was performed with a rat anti– mouse CD144/VE-cadherin (clone 11D4.1; BD Biosciences) antibody, followed by a goat anti-rat antibody conjugated with Alexa Fluor 647 (Thermo Fisher Scientific). In another set of experiments, staining of the vascular lumen was performed by administering an Alexa Fluor 594–conjugated Isolectin GS-IB 4 from Griffonia simplicifolia (Thermo Scientific), as described (75). Del-1 staining was performed with a rab- bit anti–mouse Del-1 (Proteintech, catalog 12580-1-AP), followed by a goat anti-rabbit antibody conjugated with Alexa Fluor 488 (Thermo Scientific). Nuclear staining was performed with DAPI. Images were acquired with a Zeiss LSM 510 confocal microscope, an IX83 micro- scope equipped with a Yokogawa CSU-X1 spinning disc confocal scan- ner (Olympus), or a Nikon Eclipse Ni-E automated upright fluores- cence microscope (Nikon).
Although niches have been defined for GSCs in the Drosophila ovary and testis, as well as in several tissue types of the mammalian systems, it remains unclear whether they still function properly for controlling stemcell self-renewal after their location and size are changed. In this study, we have provided two pieces of experimental evidence supporting the idea that expanded niches are functional for controlling GSC self-renewal. First, increased cap cells in the normal location express all known cap cell markers, such as hh-lacZ, wg- lacZ, Lamin C and E-cadherin, and behave like normal cap cells. Second, these expanded cap cells can support self-renewal of extra GSCs, which behave similarly to normal ones based on Dad-lacZ and bam-GFP expression, and their ability to self-renew and generate differentiated germ cells. Even when cap cells cover the anterior half of the germarium, the GSCs associated with the cap cells also appear to be capable of self-renewing and generating differentiated germ cells. Our findings show that GSC niche size can be expanded by adding more niche cells.
Fetal livers. All processing and cell enrichment procedures were conducted in a cell wash buffer composed of a basal medium (RPMI 1640) supplemented with 0.1% bovine serum albumin (BSA Fraction V, 0.1%, Sigma, St. Louis, Mo.), insulin and iron saturated transferrin both at 5 ug/ml (Sigma St Louis MO ) trace elements (selenious acid ,300 pM and ZnSO4, 50 pM), and antibiotics ( AAS, Gibco BRL/Invitrogen Corporation, Carlsbad, California). Liver tissue was subdivided into 3 mL fragments (total volume ranged from 2- 12 mL) for digestion in 25 mL of cell wash buffer containing type IV collagenase and deoxyribonuclease (Sigma Chemical Co. St Louis, both at 6 mg per mL) at 32 EC with frequent agitation for 15 – 20 minutes. This resulted in a homogeneous suspension of cell aggregates that were passed through a 40 gauge mesh and spun at 1200 RPM for five minutes before resuspension in cell wash solution. Erythrocytes were eliminated by either slow speed centrifugation (54, 55) or by treating suspensions with anti-human red blood cell (RBC) antibodies (Rockland, #109-4139) (1:5000 dilution) for 15 min followed by LowTox Guinea Pig complement (Cedarlane Labs, # CL4051) (1:3000 dilution) for 10 min both at 37°C. Estimated cell viability by trypan blue exclusion was routinely higher than 95%. See supplemental data for further details.
The stemcell microenvironment is included in directing the destiny of the The stemcell microorganism regarding self-reestablishment, calmness, and separation. Scientific models are useful in seeing how key pathways control the elements of immature microorganism upkeep and homeostasis. This tight direction and support of undeveloped cell number is thought to separate amid carcinogenesis. Therefore, the undifferentiated cell specialty has turned into a novel focus of malignancy therapeutics. Building up a quantitative comprehension of the administrative pathways that guide. The stemcellcell conduct will be key to seeing how these frameworks change under states of stress, irritation, and tumor start. Forecasts from scientific displaying can be utilized as a clinical apparatus to guide treatment outline. We introduce an overview of scientific models used to study undifferentiated organism populace elements and immature microorganism specialty direction, both in the hematopoietic framework and different tissues. Highlighting the quantitative parts of undeveloped cell science, we portray convincing inquiries that can be tended to with demonstrating. At long last, we talk about test frameworks, most remarkably Drosophila, that can best be utilized to approve scientific expectations.