Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Differential expression of endothelial protein C receptor (EPCR) in hematopoietic stem and multipotent progenitor cells in young and old mice.

Endothelial protein C receptor (EPCR) has emerged as one of the most conserved and reliable surface markers for the prospective identification and isolation of hematopoietic stem cells (HSCs). Prior studies have consistently demonstrated that EPCR expression enriches HSCs capable of long-term multilineage repopulation in both mouse and human across different hematopoietic tissues, including bone marrow (BM), fetal liver and ex vivo HSC expansion cultures. However, little is known about the expression profiles of EPCR in multipotent progenitor (MPP) populations located immediately downstream of HSCs in the hematopoietic hierarchy and which play a major role in sustaining lifelong blood cell production. Here, we incorporate EPCR antibody detection into a multi-parameter flow cytometric panel, which allows accurate identification of HSCs and five MPP subsets (MPP1-5) in mouse BM. Our data reveal that all MPP populations contain EPCR-expressing cells. Multipotent MPP1 and MPP5 contain higher proportion of EPCR+ cells compared to the more lineage-biased MPP2-4. Notably, high expression of EPCR enriches phenotypic HSC and MPP5, but not MPP1. Comparison of EPCR expression profiles between young and old BM reveals ageing mediated expansion of EPCR-expressing cells only in HSCs, but not in any of the MPP populations. Collectively, our study provides a comprehensive characterization of the surface expression pattern of EPCR in mouse HSC and MPP1-5 cells during normal and aged hematopoiesis.

Differentiation Kinetics of Hematopoietic Stem and Progenitor Cells In Vivo Are Not Affected by ß-Glucan Treatment in Trained Immunity.

Agonists of trained immunity induce epigenetic changes in hematopoietic stem and progenitor cells (HSPCs) to generate long-lasting immune protection. Although trained HSPCs generate myeloid cells with increased responsiveness to secondary challenges, whether their differentiation kinetics is affected by prior exposure to inducers of trained immunity remains elusive. Here, we used lineage tracing to examine the cell fates of endothelial protein C receptor-positive hematopoietic stem cells (EPCR+ HSCs) and fms-like tyrosine kinase 3-positive multipotent progenitor cells (Flt3+ MPPs) in ß-glucan-induced trained immunity. We found that although ß-glucan triggered the expected expansion of myeloid progenitors, the differentiation behaviors of EPCR+ HSCs and Flt3+ MPPs in multiple cycles of hematopoietic regeneration were hardly affected. Thus, our results rule out changed kinetics in cell differentiation by EPCR+ HSC and Flt3+ MPP as the cause of enhanced myelopoiesis upon secondary immune challenges.

Endothelial protein C receptor is increased in preterm preeclampsia and fetal growth restriction.

Placental dysfunction is the leading cause of both preeclampsia and fetal growth restriction. This study aimed to characterize endothelial protein C receptor (EPCR) in preterm preeclampsia, term preeclampsia, and fetal growth restriction (defined by delivery of a small for gestational age [SGA] infant [<10% birthweight centile]) and examine its regulation in primary syncytiotrophoblast. Placental EPCR mRNA and protein were significantly increased in patients with preterm preeclampsia (<34 weeks gestation) compared to gestation-matched controls (p < .0001). In the plasma, EPCR was also significantly elevated (p = .01) in established preterm preeclampsia while its substrate, protein C (PC) was significantly reduced (p = .0083). Placentas from preterm small for gestational age (SGA) cases, had elevated EPCR mRNA expression (p < .0001) relative to controls. At 36 weeks, no significant changes in plasma EPCR were detected in samples from patients destined to develop preeclampsia or deliver an SGA infant at term. In terms of syncytiotrophoblast, hypoxia significantly increased EPCR mRNA expression (p = .008), but Tumor Necrosis Factor Alpha (TNF-α) decreased EPCR mRNA. Interleukin-6 (IL-6) had no significant effect on EPCR mRNA expression. When isolated syncytiotrophoblast was treated with metformin under hypoxia (1% O2 ) or normoxia (8% O2 ), EPCR mRNA expression was significantly reduced (p = .008) relative to control. In conclusion, EPCR is markedly elevated in the placenta and the circulation of patients with established preterm preeclampsia and placental increases may be associated with hypoxia. Additionally, fetal growth-restricted pregnancies (as defined by the delivery of an SGA infant) also demonstrated elevated placental EPCR.

Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output.

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines. Irrespective of antiviral treatment or vaccination, at a certain threshold of IAV titer in the lung, CD41-positive hematopoietic stem cells (HSCs) enter the cell cycle while endothelial protein C receptor-positive CD41-negative HSCs remain quiescent. Active CD41-positive HSCs represent the source of megakaryocytes, while their multi-lineage reconstitution potential is reduced. This emergency megakaryopoiesis is thrombopoietin independent and attenuated in IAV-infected interleukin-1 receptor-deficient mice. Newly produced platelets during IAV infection are immature and hyper-reactive. After viral clearance, HSC quiescence is re-established. Our study reveals that non-systemic viral respiratory infection has an acute impact on HSCs via inflammatory cytokines to counteract IAV-induced thrombocytopenia.

Identification and characterization of in vitro expanded hematopoietic stem cells.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Factor VII, EPCR, aPC Modulators: novel treatment for neuroinflammation.

BACKGROUND: Inflammation and coagulation are linked and pathogenic in neuroinflammatory diseases. Protease-activated receptor 1 (PAR1) can be activated both by thrombin, inducing increased inflammation, and activated protein C (aPC), inducing decreased inflammation. Modulation of the aPC-PAR1 pathway may prevent the neuroinflammation associated with PAR1 over-activation. METHODS: We synthesized a group of novel molecules based on the binding site of FVII/aPC to the endothelial protein C receptor (EPCR). These molecules modulate the FVII/aPC-EPCR pathway and are therefore named FEAMs-Factor VII, EPCR, aPC Modulators. We studied the molecular and behavioral effects of a selected FEAM in neuroinflammation models in-vitro and in-vivo. RESULTS: In a lipopolysaccharide (LPS) induced in-vitro model, neuroinflammation leads to increased thrombin activity compared to control (2.7 ± 0.11 and 2.23 ± 0.13 mU/ml, respectively, p = 0.01) and decreased aPC activity (0.57 ± 0.01 and 1.00 ± 0.02, respectively, p < 0.0001). In addition, increased phosphorylated extracellular regulated kinase (pERK) (0.99 ± 0.13, 1.39 ± 0.14, control and LPS, p < 0.04) and protein kinase B (pAKT) (1.00 ± 0.09, 2.83 ± 0.81, control and LPS, p < 0.0002) levels indicate PAR1 overactivation, which leads to increased tumor necrosis factor-alpha (TNF-α) level (1.00 ± 0.04, 1.35 ± 0.12, control and LPS, p = 0.02). In a minimal traumatic brain injury (mTBI) induced neuroinflammation in-vivo model in mice, increased thrombin activity, PAR1 activation, and TNF-α levels were measured. Additionally, significant memory impairment, as indicated by a lower recognition index in the Novel Object Recognition (NOR) test and Y-maze test (NOR: 0.19 ± 0.06, -0.07 ± 0.09, p = 0.03. Y-Maze: 0.50 ± 0.03, 0.23 ± 0.09, p = 0.02 control and mTBI, respectively), as well as hypersensitivity by hot-plate latency (16.6 ± 0.89, 12.8 ± 0.56 s, control and mTBI, p = 0.01), were seen. FEAM prevented most of the molecular and behavioral negative effects of neuroinflammation in-vitro and in-vivo, most likely through EPCR-PAR1 interactions. CONCLUSION: FEAM is a promising tool to study neuroinflammation and a potential treatment for a variety of neuroinflammatory diseases.

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.

ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration.

In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5-fluorouracil-induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis-regulatory elements in progenitor cell-related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY-dependent histone acetylation promoted differentiation into CD48+ progenitor cells. Collectively, these results indicate that the dynamic control of ACLY-dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.

Protein C receptor maintains cancer stem cell properties via activating lipid synthesis in nasopharyngeal carcinoma.

Metastasis and recurrence account for 95% of deaths from nasopharyngeal carcinoma (NPC). Cancer stem cells (CSCs) are regarded as one of the main reasons for tumor cell resistance to clinical therapy, and cancer metastasis or recurrence, while little is known about CSCs in NPC. The present study uncovers a subpopulation of cells labeled as CD45-EPCAM+PROCR+ in NPC biopsy samples that exhibit stem cell-like characteristics. A relatively low number of these cells initiate xenograft tumors in mice. Functional analysis reveals that protein C receptor (PROCR) not only serves as a stem cell marker in NPC, but also maintains tumor cells' stemness potential through regulating lipid metabolism and mitochondrial fission. Epistatic studies reveal that cAMP-protein kinase A stimulates Ca2+ release to manipulate lipid metabolism related genes' expression. Finally, in a cohort of 207 NPC samples, PROCR expression is correlated with tumor metastasis or recurrence, and predicts poor prognosis. These novel findings link PROCR labeled CSCs with lipid metabolism and mitochondrial plasticity, and provides new clinical target against metastatic or recurrent NPC.

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis.

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.

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.

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes.

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1-/- mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.

A critical role of endothelial cell protein C receptor in the intestinal homeostasis in experimental colitis.

Crohn's disease and ulcerative colitis are the two forms of disorders of the human inflammatory bowel disease with unknown etiologies. Endothelial cell protein C receptor (EPCR) is a multifunctional and multiligand receptor, which is expressed on the endothelium and other cell types, including epithelial cells. Here, we report that EPCR is expressed in the colon epithelial cells, CD11c+, and CD21+/CD35+ myeloid cells surrounding the crypts in the colon mucosa. EPCR expression was markedly decreased in the colon mucosa during colitis. The loss of EPCR appeared to associate with increased disease index of the experimental colitis in mice. EPCR-/- mice were more susceptible to dextran sulfate sodium (DSS)-induced colitis, manifested by increased weight loss, macrophage infiltration, and inflammatory cytokines in the colon tissue. DSS treatment of EPCR-/- mice resulted in increased bleeding, bodyweight loss, anemia, fibrin deposition, and loss of colon epithelial and goblet cells. Administration of coagulant factor VIIa significantly attenuated the DSS-induced colon length shortening, rectal bleeding, bodyweight loss, and disease activity index in the wild-type mice but not EPCR-/- mice. In summary, our data provide direct evidence that EPCR plays a crucial role in regulating the inflammation in the colon during colitis.

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.

Evolution of ASC Immunophenotypical Subsets During Expansion In Vitro.

Adipose-derived stromal/stem cells (ASCs) are currently being considered for clinical use for a number of indications. In order to develop standardized clinical protocols, it is paramount to have a full characterization of the stem cell preparations. The surface marker expression of ASCs has previously been characterized in multiple studies. However, most of these studies have provided a cross-sectional description of ASCs in either earlier or later passages. In this study, we evaluate the dynamic changes of 15 different surface molecules during culture. Using multichromatic flow cytometry, ASCs from three different donors each in passages 1, 2, 4, 6, and 8 were analyzed for their co-expression of markers associated with mesenchymal stem cells, wound healing, immune regulation, ASC markers, and differentiation capacity, respectively. We confirmed that at an early stage, ASC displayed a high heterogeneity with a plethora of subpopulations, which by culturing became more homogeneous. After a few passages, virtually all ASCs expressed CD29, CD166 and CD201, in addition to canonical markers CD73, CD90, and CD105. However, even at passage 8, there were several predominant lineages that differed with respect to the expression of CD34, CD200 and CD271. Although the significance of remaining subpopulations still needs to be elucidated, our results underscore the necessity to fully characterize ASCs prior to clinical use.

CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.

Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR+/CD34- fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress.

Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan.

The functional heterogeneity of hematopoietic stem cells (HSCs) has been comprehensively investigated by single-cell transplantation assay. However, the heterogeneity regarding their physiological contribution remains an open question, especially for those with life-long hematopoietic fate of rigorous self-renewing and balanced differentiation capacities. In this study, we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing Dll4 and CD44 in the mid-gestation mouse embryos, and could enrich all the HSCs of the embryonic day 11.5 (E11.5) aorta-gonad-mesonephros (AGM) region. We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-expressing cells at E9.5. Interestingly, most labeled mature HSCs in multiple sites (such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation. Furthermore, the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver (FL) and 6.9% of leukocytes in peripheral blood (PB) during one-year follow-up. Surprisingly, in aged mice of 24 months, the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages. Altogether, we demonstrated, for the first time, the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized, whose precise embryonic origin and molecular identity await further characterization.

Procr-expressing progenitor cells are responsible for murine ovulatory rupture repair of ovarian surface epithelium.

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and repair. The OSE replenishing mechanism post ovulation remains unclear. Here we report that the expression of Protein C Receptor (Procr) marks a progenitor population in adult mice that is responsible for OSE repair post ovulation. Procr+ cells are the major cell source for OSE repair. The mechanism facilitating the rapid re-epithelialization is through the immediate expansion of Procr+ cells upon OSE rupture. Targeted ablation of Procr+ cells impedes the repairing process. Moreover, Procr+ cells displayed robust colony-formation capacity in culture, which we harnessed and established a long-term culture and expansion system of OSE cells. Finally, we show that Procr+ cells and previously reported Lgr5+ cells have distinct lineage tracing behavior in OSE homeostasis. Our study suggests that Procr marks progenitor cells that are critical for OSE ovulatory rupture and homeostasis, providing insight into how adult stem cells respond upon injury.

Protein C receptor is a therapeutic stem cell target in a distinct group of breast cancers.

Breast cancer is a heterogeneous disease. In particular, triple-negative breast cancer (TNBC) comprises various molecular subgroups with unclear identities and currently has few targeted treatment options. Our previous study identified protein C receptor (Procr) as a surface marker on mammary stem cells (MaSCs) located in the basal layer of the normal mammary gland. Given the possible connection of TNBC with basal layer stem cells, we conducted comparative analyses of Procr in breast cancers of mouse and human origin. In mouse mammary tumors, we showed that Procr+ cells are enriched for cancer stem cells (CSCs) in Wnt1 basal-like tumors, but not in Brca1 basal-like tumors or PyVT luminal tumors. In human cancers, PROCR was robustly expressed in half of TNBC cases. Experiments with patient-derived xenografts (PDXs) revealed that PROCR marks CSCs in this discrete subgroup (referred to as PROCR+ TNBC). Interfering with the function of PROCR using an inhibitory nanobody reduced the CSC numbers, arrested tumor growth and prevented rapid tumor recurrence. Our data suggest a key role of MaSC in breast tumorigenesis. Moreover, our work indicates that PROCR can be used as a biomarker to stratify TNBC into clinically relevant subgroups and may provide a novel targeted treatment strategy for this clinically important tumor subtype.