Long-Term Expansion of Pancreatic Islet Organoids from Resident Procr+ Progenitors.

It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing ∼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.

MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.

Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.

R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal.

Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/ß-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.

Amphiregulin mediates the hormonal regulation on Rspondin-1 expression in the mammary gland.

The steroid hormones are instrumental for the growth of mammary epithelial cells. Our previous study indicates that hormones regulate the expression of Rspondin-1 (Rspo1). Yet, the regulatory mechanism remains unknown. In the current study, we identify Amphiregulin (Areg) as a novel upstream regulator of Rspo1 expression mediating the hormonal influence. In response to hormonal signaling, Areg emanating from estrogen receptor (ER)-positive luminal cells, induce the expression of Rspo1 in ER-negative luminal cells. The paracrine action of Areg on Rspo1 expression is dependent on Egfr. Our data reveal a novel Estrogen-Areg-Rspo1 regulatory axis in the mammary gland, providing new evidence for the orchestrated action of systemic hormones and local growth factors.

Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium.

BACKGROUND: Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/ß-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins. RESULTS: We established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDß (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression. CONCLUSION: We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.

Identification of multipotent mammary stem cells by protein C receptor expression.

The mammary gland is composed of multiple types of epithelial cells, which are generated by mammary stem cells (MaSCs) residing at the top of the hierarchy. However, the existence of these multipotent MaSCs remains controversial and the nature of such cells is unknown. Here we demonstrate that protein C receptor (Procr), a novel Wnt target in the mammary gland, marks a unique population of multipotent mouse MaSCs. Procr-positive cells localize to the basal layer, exhibit epithelial-to-mesenchymal transition characteristics, and express low levels of basal keratins. Procr-expressing cells have a high regenerative capacity in transplantation assays and differentiate into all lineages of the mammary epithelium by lineage tracing. These results define a novel multipotent mammary stem cell population that could be important in the initiation of breast cancer.

Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.

The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.

A mutation that blocks integrin α4ß7 activation prevents adaptive immune-mediated colitis without increasing susceptibility to innate colitis.

BACKGROUND: ß7 integrins are responsible for the efficient recruitment of lymphocytes from the blood and their retention in gut-associated lymphoid tissues. Integrin α4ß7 binds MAdCAM-1, mediating rolling adhesion of lymphocytes on blood vessel walls when inactive and firm adhesion when activated, thereby controlling two critical steps of lymphocyte homing to the gut. By contrast, integrin αEß7 mediates the adhesion of lymphocytes to gut epithelial cells by interacting with E-cadherin. Integrin ß7 blocking antibodies have shown efficacy in clinical management of inflammatory bowel disease (IBD); however, fully blocking ß7 function leads to the depletion of colonic regulatory T (Treg) cells and exacerbates dextran sulfate sodium (DSS)-induced colitis by evoking aberrant innate immunity, implying its potential adverse effect for IBD management. Thus, a better therapeutic strategy targeting integrin ß7 is required to avoid this adverse effect. RESULTS: Herein, we inhibited integrin α4ß7 activation in vivo by creating mice that carry in their integrin ß7 gene a mutation (F185A) which from structural studies is known to lock α4ß7 in its resting state. Lymphocytes from ß7-F185A knock-in (KI) mice expressed α4ß7 integrins that could not be activated by chemokines and showed significantly impaired homing to the gut. The ß7-F185A mutation did not inhibit αEß7 activation, but led to the depletion of αEß7+ lymphocytes in the spleen and a significantly reduced population of αEß7+ lymphocytes in the gut of KI mice. ß7-F185A KI mice were resistant to T cell transfer-induced chronic colitis, but did not show an increased susceptibility to DSS-induced innate colitis, the adverse effect of fully blocking ß7 function. CONCLUSIONS: Our findings demonstrate that specific inhibition of integrin α4ß7 activation is a potentially better strategy than fully blocking α4ß7 function for IBD treatment.

Protein C receptor stimulates multiple signaling pathways in breast cancer cells.

The protein C receptor (PROCR) has emerged as a stem cell marker in several normal tissues and has also been implicated in tumor progression. However, the functional role of PROCR and the signaling mechanisms downstream of PROCR remain poorly understood. Here, we dissected the PROCR signaling pathways in breast cancer cells. Combining protein array, knockdown, and overexpression methods, we found that PROCR concomitantly activates multiple pathways. We also noted that PROCR-dependent ERK and PI3k-Akt-mTOR signaling pathways proceed through Src kinase and transactivation of insulin-like growth factor 1 receptor (IGF-1R). These pathway activities led to the accumulation of c-Myc and cyclin D1. On the other hand, PROCR-dependent RhoA-ROCK-p38 signaling relied on coagulation factor II thrombin receptor (F2R). We confirmed these findings in primary cells isolated from triple-negative breast cancer-derived xenografts (PDX) that have high expression of PROCR. To the best our knowledge, this is the first comprehensive study of PROCR signaling in breast cancer cells, and its findings also shed light on the molecular mechanisms of PROCR in stem cells in normal tissue.

Essential Roles of Cyclin Y-Like 1 and Cyclin Y in Dividing Wnt-Responsive Mammary Stem/Progenitor Cells.

Cyclin Y family can enhance Wnt/ß-catenin signaling in mitosis. Their physiological roles in mammalian development are yet unknown. Here we show that Cyclin Y-like 1 (Ccnyl1) and Cyclin Y (Ccny) have overlapping function and are crucial for mouse embryonic development and mammary stem/progenitor cell functions. Double knockout of Ccnys results in embryonic lethality at E16.5. In pubertal development, mammary terminal end buds robustly express Ccnyl1. Depletion of Ccnys leads to reduction of Lrp6 phosphorylation, hampering ß-catenin activities and abolishing mammary stem/progenitor cell expansion in vitro. In lineage tracing experiments, Ccnys-deficient mammary cells lose their competitiveness and cease to contribute to mammary development. In transplantation assays, Ccnys-deficient mammary cells fail to reconstitute, whereas constitutively active ß-catenin restores their regeneration abilities. Together, our results demonstrate the physiological significance of Ccnys-mediated mitotic Wnt signaling in embryonic development and mammary stem/progenitor cells, and reveal insights in the molecular mechanisms orchestrating cell cycle progression and maintenance of stem cell properties.

Tympanic border cells are Wnt-responsive and can act as progenitors for postnatal mouse cochlear cells.

Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.

Niche inflammatory signals control oscillating mammary regeneration and protect stem cells from cytotoxic stress.

Stem cells are known for their resilience and enhanced activity post-stress. The mammary gland undergoes frequent remodeling and is subjected to recurring stress during the estrus cycle, but it remains unclear how mammary stem cells (MaSCs) respond to the stress and contribute to regeneration. We discovered that cytotoxic stress-induced activation of CD11c+ ductal macrophages aids stem cell survival and prevents differentiation. These macrophages boost Procr+ MaSC activity through IL1ß-IL1R1-NF-κB signaling during the estrus cycle in an oscillating manner. Deleting IL1R1 in MaSCs results in stem cell loss and skewed luminal differentiation. Moreover, under cytotoxic stress from the chemotherapy agent paclitaxel, ductal macrophages secrete higher IL1ß levels, promoting MaSC survival and preventing differentiation. Inhibiting IL1R1 sensitizes MaSCs to paclitaxel. Our findings reveal a recurring inflammatory process that regulates regeneration, providing insights into stress-induced inflammation and its impact on stem cell survival, potentially affecting cancer therapy efficacy.

Ca2+ transients on the T cell surface trigger rapid integrin activation in a timescale of seconds.

One question in lymphocyte homing is how integrins are rapidly activated to enable immediate arrest of fast rolling lymphocytes upon encountering chemokines at target vascular beds given the slow chemokine-induced integrin inside-out activation. Herein we demonstrate that chemokine CCL25-triggered Ca2+ influx induces T cell membrane-proximal external Ca2+ concentration ([Ca2+]ex) drop in 6 s from physiological concentration 1.2 mM to 0.3 mM, a critical extracellular Ca2+ threshold for inducing αLß2 activation, triggering rapid αLß2 activation and T cell arrest before occurrence of αLß2 inside-out activation. Talin knockdown inhibits the slow inside-out activation of αLß2 but not [Ca2+]ex drop-triggered αLß2 quick activation. Blocking Ca2+ influx significantly suppresses T cell rolling-to-arrest transition and homing to skin lesions in a mouse psoriasis model, thus alleviating skin inflammation. [Ca2+]ex decrease-triggered rapid integrin activation bridges the gap between initial chemokine stimulation and slow integrin inside-out activation, ensuring immediate lymphocyte arrest and subsequent diapedesis on the right location.

LRP12 is an endogenous transmembrane inactivator of α4 integrins.

Dynamic regulation of integrin activation and inactivation is critical for precisely controlled cell adhesion and migration in physiological and pathological processes. The molecular basis for integrin activation has been intensively studied; however, the understanding of integrin inactivation is still limited. Here, we identify LRP12 as an endogenous transmembrane inhibitor for α4 integrin activation. The LRP12 cytoplasmic domain directly binds to the integrin α4 cytoplasmic tail and inhibits talin binding to the ß subunit, thus keeping integrin inactive. In migrating cells, LRP12-α4 interaction induces nascent adhesion (NA) turnover at the leading-edge protrusion. Knockdown of LRP12 leads to increased NAs and enhanced cell migration. Consistently, LRP12-deficient T cells show an enhanced homing capability in mice and lead to aggravated chronic colitis in a T cell-transfer colitis model. Altogether, LRP12 is a transmembrane inactivator for integrins that inhibits α4 integrin activation and controls cell migration by maintaining balanced NA dynamics.

Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet-Induced Adiposity.

Recent genetic evidence has linked WNT downstream mutations to fat distribution. However, the roles of WNTs in human obesity remain unclear. Here, the authors screen all Wnt-related paracrine factors in 1994 obese cases and 2161 controls using whole-exome sequencing (WES) and identify that 12 obese patients harbor the same mutations in RSPO1 (p.R219W/Q) predisposing to human obesity. RSPO1 is predominantly expressed in visceral fat, primarily in the fibroblast cluster, and is increased with adiposity. Mice overexpressing human RSPO1 in adipose tissues develop obesity under a high-fat diet (HFD) due to reduced brown/beige fat thermogenesis. In contrast, Rspo1 ablation resists HFD-induced adiposity by increasing thermogenesis. Mechanistically, RSPO1 overexpression or administration significantly inhibits adipocyte mitochondrial respiration and thermogenesis via LGR4-Wnt/ß-catenin signaling pathway. Importantly, humanized knockin mice carrying the hotspot mutation (p.R219W) display suppressed thermogenesis and recapitulate the adiposity feature of obese carriers. The mutation disrupts RSPO1's electrostatic interaction with the extracellular matrix, leading to excessive RSPO1 release that activates LGR4-Wnt/ß-catenin signaling and attenuates thermogenic capacity in differentiated beige adipocytes. Therefore, these findings identify that gain-of-function mutations and excessive expression of RSPO1, acting as a paracrine Wnt activator, suppress fat thermogenesis and contribute to obesity in humans.

Isolation of mouse pancreatic islet Procr+ progenitors and long-term expansion of islet organoids in vitro.

Insulin production is required for glucose homeostasis. Pancreatic islet ß cells are the only cells that produce insulin in humans; however, generation of functional ß cells in vitro from embryonic or adult tissues has been challenging. Here, we describe isolation of pancreatic islet progenitors from adult mice, which enables the efficient generation and long-term expansion of functional islet organoids in vitro. This protocol starts with purification of protein C receptor (Procr)-expressing islet progenitors. Coculture with endothelial cells generates islet organoids in vitro that can be expanded by passage. Functional maturation is achieved as a consequence of a prolonged culture period and cyclic glucose stimulation. Primary islet organoids form in 7-10 days. Subsequently, each passage takes 1 week, with the final maturation step requiring 3 weeks of additional culture. The resulting organoids are predominantly composed of ß cells but also contain small proportions of α, δ and pancreatic polypeptide cells. The organoids sense glucose and secrete insulin. This approach thus provides a strategy for ß cell generation in vitro and an organoid system to study islet regeneration and diseases.

Procr functions as a signaling receptor and is essential for the maintenance and self-renewal of mammary stem cells.

The protein C receptor (Procr) has been implicated as a stem cell surface marker in several tissues. It is unknown whether Procr acts as a functional signaling receptor in stem cells. Here, by conditional knockout in mammary stem cells (MaSCs), we demonstrate that Procr is essential for mammary gland development and homeostasis. Through proteomics profiling, we identify that, upon stimulation by the ligand protein C, Procr interacts with heat shock protein 90 (HSP90AA1) via its short cytoplasmic tail, recruiting Src and IGF1R to the complex at the plasma membrane. We show that Procr acts as a signaling receptor of protein C in regulation of MaSCs through HSP90, Src, and IGF1R in vitro. In vivo, IGF1R deletion in MaSCs displays similar phenotypes to Procr deletion. These findings illustrate the essential role of Procr signaling in MaSC maintenance, shedding light onto the molecular regulation by Procr in tissue stem cells.

CDK14 inhibition reduces mammary stem cell activity and suppresses triple negative breast cancer progression.

The Wnt/ß-catenin signaling pathway plays an important role in regulating mammary organogenesis and oncogenesis. However, therapeutic methods targeting the Wnt pathway against breast cancer have been limited. To address this challenge, we investigate the function of cyclin-dependent kinase 14 (CDK14), a member of the Wnt signaling pathway, in mammary development and breast cancer progression. We show that CDK14 is expressed in the mammary basal layer and elevated in triple negative breast cancer (TNBC). CDK14 knockdown reduces the colony-formation ability and regeneration capacity of mammary basal cells and inhibits the progression of murine MMTV-Wnt-1 basal-like mammary tumor. CDK14 knockdown or pharmacological inhibition by FMF-04-159-2 suppresses the progression and metastasis of TNBC. Mechanistically, CDK14 inhibition inhibits mammary regeneration and TNBC progression by attenuating Wnt/ß-catenin signaling. These findings highlight the significance of CDK14 in mammary development and TNBC progression, shedding light on CDK14 as a promising therapeutic target for TNBC.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair.

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell activation is triggered by the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap1 in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP-dependent mechanism by which the stem/progenitor cells recognize the murine ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP-induced stem cell expansion strategy.