FLT3LG and IFITM3P6 consolidate T cell activity in the bone marrow microenvironment and are prognostic factors in acute myelocytic leukemia.

Acute myelocytic leukemia (AML) is a malignancy of the stem cell precursors of the myeloid lineage. CD4+ and CD8+ T cells play pivotal roles in influencing AML progression but are functionally suppressed in the bone marrow microenvironment. We aimed to find hub genes related to T cell exhaustion and suppression, thereby providing evidence for immunotherapy. In this study, gene transcriptome expression data from TCGA and TARGET databases were utilized to find key genes. Firstly, CIBERSORT immune cell infiltration algorithm and WGCNA method were used to identify CD4+ and CD8+ T cells-related genes. Univariate and multivariate cox regression analyses were then introduced to construct the overall survival prognosis model and included hub genes. The ESTIMATE and ssGSEA scoring methods were used to analyze the correlation between the hub genes and immune activity. Single-cell transcriptome analysis was applied to detect the immune cells expressing hub genes, hence, to detect exact mechanisms. Consequently, FLT3LG and IFITM3P6 were determined to be positively correlated with patients' overall survival and microenvironment immune activity. Further study suggested FLT3-FLT3LG and IFITM3P6-miR-6748-3p-CBX7 signaling axes were involved in CD4+ and CD8+ T cells activation. This may be one of the mechanisms of T cells suppression in AML.

Daidzein Activates Akt Pathway to Promote the Proliferation of Female Germline Stem Cells through Upregulating Clec11a.

Female germline stem cells (FGSCs) have been successfully isolated and characterized from postnatal mammalian and human ovarian tissues. However, the effects and mechanisms of action of natural small-molecule compounds on FGSCs are largely unknown. Here, we found that daidzein promoted the viability and proliferation of FGSCs. To elucidate the mechanism underlying this, we performed RNA-Sequence in daidzein-treated FGSCs and controls. The results showed that there were 153 upregulated and 156 downregulated genes in daidzein treatment. We confirmed the expression of some genes related to cell proliferation in the sequencing results by RT-PCR, such as Type C lectin domain family 11 member a (Clec11a), Mucin1 (Muc1), Glutathione peroxidase 3 (Gpx3), and Tet methylcytosine dioxygenase 1 (Tet1). The high expression of Clec11a at the protein level after daidzein treatment was also confirmed by western blotting. Furthermore, recombinant mouse Clec11a (rmClec11a) protein was shown to promote the viability and proliferation of FGSCs. However, knockdown of Clec11a inhibited the viability and proliferation of FGSCs, which could not be rescued by the administration of daidzein. These results indicate that daidzein promoted the viability and proliferation of FGSCs through Clec11a. In addition, both daidzein and rmClec11a activated the Akt signaling pathway in FGSCs. However, Clec11a knockdown inhibited this pathway, which could not be rescued by daidzein administration. Taken together, our findings revealed that daidzein activates the Akt signaling pathway to promote cell viability and proliferation through upregulating Clec11a. This study should deepen our understanding of the developmental mechanism of FGSCs and female infertility.

NCCN Guidelines® Insights: Hematopoietic Growth Factors, Version 1.2022.

The NCCN Guidelines for Hematopoietic Growth Factors provide recommendations for the appropriate use of growth factors in the clinical management of febrile neutropenia (FN), chemotherapy-induced thrombocytopenia (CIT), and chemotherapy-induced anemia (CIA). Management and prevention of these sequelae are an integral part of supportive care for many patients undergoing cancer treatment. The purpose of these guidelines is to operationalize the evaluation, prevention, and treatment of FN, CIT, and CIA in adult patients with nonmyeloid malignancies and to enable the patient and clinician to assess management options for FN, CIT, and CIA in the context of an individual patient's condition. These NCCN Guidelines Insights provide a summary of the important recent updates to the NCCN Guidelines for Hematopoietic Growth Factors, with particular emphasis on the incorporation of a newly developed section on CIT.

Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks.

The systemic processes involved in the manifestation of life-threatening COVID-19 and in disease recovery are still incompletely understood, despite investigations focusing on the dysregulation of immune responses after SARS-CoV-2 infection. To define hallmarks of severe COVID-19 in acute disease (n = 58) and in disease recovery in convalescent patients (n = 28) from Hannover Medical School, we used flow cytometry and proteomics data with unsupervised clustering analyses. In our observational study, we combined analyses of immune cells and cytokine/chemokine networks with endothelial activation and injury. ICU patients displayed an altered immune signature with prolonged lymphopenia but the expansion of granulocytes and plasmablasts along with activated and terminally differentiated T and NK cells and high levels of SARS-CoV-2-specific antibodies. The core signature of seven plasma proteins revealed a highly inflammatory microenvironment in addition to endothelial injury in severe COVID-19. Changes within this signature were associated with either disease progression or recovery. In summary, our data suggest that besides a strong inflammatory response, severe COVID-19 is driven by endothelial activation and barrier disruption, whereby recovery depends on the regeneration of the endothelial integrity.

The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin.

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor+ (LepR+) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

Measurement of multiple cytokines for discrimination and risk stratification in patients with Chagas' disease and idiopathic dilated cardiomyopathy.

Chagas' disease (CD), caused by the hemoflagellate protozoan, Trypanosoma cruzi, is endemic in most countries of Latin America. Heart failure (HF) is often a late manifestation of chronic CD, and is associated with high morbidity and mortality. Inflammatory processes mediated by cytokines play a key role in the pathogenesis and progression of CD. Keeping in view the inflammatory nature of CD, this study investigated the possible role of 21 different inflammatory cytokines as biomarkers for prediction and prognosis of CD. The plasma concentration of these cytokines was measured in a group of patients with CD (n = 94), and then compared with those measured in patients with dilated cardiomyopathy (DCM) from idiopathic causes (n = 48), and with control subjects (n = 25). Monovariately, plasma levels of cytokines such as stem cell growth factor beta (SCGF beta), hepatocyte growth factor (HGF), monokine induced by interferon gamma (CXCL9), and macrophage inhibitory factor (MIF) were significantly increased in CD patients with advanced HF compared to control group. None of the cytokines could demonstrate any prognostic potency in CD patients, and only MIF and stromal derived factor-1 alpha (CXCL12) showed significance in predicting mortality and necessity for heart transplant in DCM patients. However, multivariate analysis prognosticated a large proportion of CD and DCM patients. In CD patients, HGF and Interleukin-12p40 (IL-12p40) together separated 81.9% of 3-year survivors from the deceased, while in DCM patients, CXCL12, stem cell factor (SCF), and CXCL9 together discriminated 77.1% of survivors from the deceased. The significant increase in plasma concentrations of cytokines such as HGF and CXCL9 in CD patients, and the ability of these cytokines to prognosticate a large proportion of CD and DCM patients multivariately, encourages further studies to clarify the diagnostic and prognostic potential of cytokines in such patients.

Stem cell-secreted factor therapy regenerates the ovarian niche and rescues follicles.

BACKGROUND: Ovarian senescence is a normal age-associated phenomenon, but increasingly younger women are affected by diminished ovarian reserves or premature ovarian insufficiency. There is an urgent need for developing therapies to improve ovarian function in these patients. In this context, previous studies suggest that stem cell-secreted factors could have regenerative properties in the ovaries. OBJECTIVE: This study aimed to test the ability of various human plasma sources, enriched in stem cell-secreted factors, and the mechanisms behind their regenerative properties, to repair ovarian damage and to promote follicular development. STUDY DESIGN: In the first phase, the effects of human plasma enriched in bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization, umbilical cord blood plasma, and their activated forms on ovarian niche, follicle development, and breeding performance were assessed in mouse models of chemotherapy-induced ovarian damage (n=7 per group). In addition, the proteomic profile of each plasma was analyzed to find putative proteins and mechanism involved in their regenerative properties in ovarian tissue. In the second phase, the most effective plasma treatment was validated in human ovarian cortex xenografted in immunodeficient mice (n=4 per group). RESULTS: Infusion of human plasma enriched bone marrow stem cell soluble factors by granulocyte colony-stimulating factor mobilization or of umbilical cord blood plasma-induced varying degrees of microvessel formation and cell proliferation and reduced apoptosis in ovarian tissue to rescue follicular development and fertility in mouse models of ovarian damage. Plasma activation enhanced these effects. Activated granulocyte colony-stimulating factor plasma was the most potent inducing ovarian rescue in both mice and human ovaries, and proteomic analysis indicated that its effects may be mediated by soluble factors related to cell cycle and apoptosis, gene expression, signal transduction, cell communication, response to stress, and DNA repair of double-strand breaks, the most common form of age-induced damage in oocytes. CONCLUSION: Our findings suggested that stem cell-secreted factors present in both granulocyte colony-stimulating factor-mobilized and umbilical cord blood plasma could be an effective treatment for increasing the reproductive outcomes in women with impaired ovarian function owing to several causes. The activated granulocyte colony-stimulating factor plasma, which is already enriched in both stem cell-secreted factors and platelet-enclosed growth factors, seems to be the most promising treatment because of its most potent restorative effects on the ovary together with the autologous source.

A mechanosensitive peri-arteriolar niche for osteogenesis and lymphopoiesis.

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors1-6. LEPR+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin13,14, distinguishes peri-arteriolar LEPR+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR+osteolectin+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin+ cells depleted osteolectin+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing.

Use of molecularly-cloned haematopoietic growth factors in persons exposed to acute high-dose, high-dose rate whole-body ionizing radiations.

Exposure to acute, high-dose, high dose-rate whole-body ionizing radiations damages the bone marrow resulting in rapid decreases in concentrations of blood cells, especially lymphocytes, granulocytes and platelets with associated risks of infection and bleeding. In several experimental models including non-human primate radiation exposure models giving molecularly cloned haematopoietic growth factor including granulocyte/macrophage colony-stimulating factor (G/M-CSF; sargramostim) and granulocyte colony-stimulating factor (G-CSF; filgrastim and pegylated G-CSF [peg-filgrastim]) accelerates bone marrow recovery and increases survival. Based on these data these molecules are US FDA approved for treating victims of radiation and nuclear incidents, accident and events such as nuclear terrorism and are included in the US National Strategic Stockpile. We discuss the immediate medical response to these events including how to estimate radiation dose and uniformity and which interventions are appropriate in different radiation exposures settings. We also discuss similarities and differences between molecularly cloned haematopoietic growth factors.

Identification of Fibroblast Activation Protein as an Osteogenic Suppressor and Anti-osteoporosis Drug Target.

Osteogenic suppressors such as Sclerostin not only regulate skeletal development and regeneration but also serve as anti-osteoporosis drug targets. However, very few druggable suppressors have been identified due to limited understanding of the molecular mechanisms governing osteogenesis. Here, we show that fibroblast activation protein (Fap), a serine protease inhibited by the bone growth factor Osteolectin, is an osteogenic suppressor. Genetic deletion of Fap significantly ameliorates limb trabecular bone loss during aging. Pharmacological inhibition of Fap significantly promotes bone formation and inhibits bone resorption in wild-type mice by differentially regulating canonical Wnt and nuclear factor κB (NF-κB) pathways. Pharmacological inhibition of Fap promotes osteoblast differentiation, inhibits osteoclast differentiation, and significantly attenuates osteoporosis in ovariectomized mice. Epistasis analyses in zebrafish show that Osteolectin functions as an endogenous inhibitor of Fap to promote vertebrae mineralization. Taken together, we identify Fap as an important osteogenic suppressor and a potential drug target to treat osteoporosis.

Dnmt3a-null hematopoietic stem and progenitor cells expand after busulfan treatment.

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a-/- HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of ∼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.

Molecular structure, expression, and functional role of Clec11a in skeletal biology and cancers.

C-type lectin domain family 11 member A (Clec11a), also known as stem cell growth factor (SCGF), C-type lectin superfamily member 3 (CLECSF3), or osteolectin was initially identified as a growth factor for hematopoietic progenitor cells. The human Clec11a gene encodes a polypeptide of 323 amino acids with characteristics of a secreted glycoprotein encompassing two integrin-binding motifs, RGD (Arg-Gly-Asp) and LDT (Leu-Asp-Thr), a putative leucine zipper domain, and a functional C-type lectin domain. It regulates hematopoietic differentiation and homeostasis and exhibits a protective effect against severe malarial anemia and lipotoxicity. Furthermore, Clec11a promotes the differentiation of mesenchymal progenitors into mature osteoblasts in vitro and plays an important role in the maintenance of adult skeleton age-related bone loss and fracture repair. Receptor ligand binding results in activation of downstream signaling cascades including glycogen synthase kinase 3 (GSK3), ß-catenin, and Wnt, resulting in the expression of osteoblast-related gene transcripts including Alp, Runx2, Lef1, and Axin2. In addition, Clec11a is also associated with the development of several cancers, including leukemia, multiple myeloma, and gastrointestinal tract tumors. To date, however, the mechanisms governing transcription regulation of the Clec11a gene are not known and remain to be uncovered. Understanding the function and mechanism of action of Clec11a will pave the way for the development of Clec11a as a novel therapeutic target for conditions such as cancer, anemia, and skeletal diseases.

Chronic activation of endothelial MAPK disrupts hematopoiesis via NFKB dependent inflammatory stress reversible by SCGF.

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.

Human umbilical cord mesenchymal stromal cells-derived extracellular vesicles exert potent bone protective effects by CLEC11A-mediated regulation of bone metabolism.

Osteoporosis and osteoporotic fractures severely compromise quality of life in elderly people and lead to early death. Human umbilical cord mesenchymal stromal cell (MSC)-derived extracellular vesicles (hucMSC-EVs) possess considerable therapeutic effects in tissue repair and regeneration. Thus, in the present study, we investigated the effects of hucMSC-EVs on primary and secondary osteoporosis and explored the underlying mechanisms. Methods: hucMSCs were isolated and cultured. EVs were obtained from the conditioned medium of hucMSCs and determined by using transmission electron microscopy, dynamic light scattering and Western Blot analyses. The effects of hucMSC-EVs on ovariectomy-induced postmenopausal osteoporosis and tail suspension-induced hindlimb disuse osteoporosis in mouse models were assessed by using microcomputed tomography, biomechanical, histochemical and immunohistochemical, as well as histomorphometric analyses. Proteomic analysis was applied between hucMSC-EVs and hucMSCs to screen the candidate proteins that mediate hucMSC-EVs function. The effects of hucMSC-EVs on osteogenic and adipogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), and osteoclastogenesis of the macrophage cell line RAW264.7 in vitro were determined by using cytochemical staining and quantitative real-time PCR analysis. Subsequently, the roles of the key protein in hucMSC-EVs-induced regulation on BMSCs and RAW264.7 cells were evaluated. Results: hucMSCs were able to differentiate into osteoblasts, adipocytes or chondrocytes and positively expressed CD29, CD44, CD73 and CD90, but negatively expressed CD34 and CD45. The morphological assessment revealed the typical cup- or sphere-shaped morphology of hucMSC-EVs with diameters predominantly ranging from 60 nm to 150 nm and expressed CD9, CD63, CD81 and TSG101. The systemic administration of hucMSC-EVs prevented bone loss and maintained bone strength in osteoporotic mice by enhancing bone formation, reducing marrow fat accumulation and decreasing bone resorption. Proteomic analysis showed that the potently pro-osteogenic protein, CLEC11A (C-type lectin domain family 11, member A) was very highly enriched in hucMSC-EVs. In addition, hucMSC-EVs enhanced the shift from adipogenic to osteogenic differentiation of BMSCs via delivering CLEC11A in vitro. Moreover, CLEC11A was required for the inhibitory effects of hucMSC-EVs on osteoclast formation. Conclusion: Our results suggest that hucMSC-EVs serve as a critical regulator of bone metabolism by transferring CLEC11A and may represent a potential agent for prevention and treatment of osteoporosis.

NCCN Guidelines Insights: Hematopoietic Growth Factors, Version 1.2020.

Management of febrile neutropenia (FN) is an integral part of supportive care for patients undergoing cancer treatment. The NCCN Guidelines for Hematopoietic Growth Factors provide suggestions for appropriate evaluation, risk determination, prophylaxis, and management of FN. These NCCN Guidelines are intended to guide clinicians in the appropriate use of growth factors for select patients undergoing treatment of nonmyeloid malignancies. These NCCN Guidelines Insights highlight important updates to the NCCN Guidelines regarding the incorporation of newly FDA-approved granulocyte-colony stimulating factor biosimilars for the prevention and treatment of FN.

Normal Hematopoiesis Is a Balancing Act of Self-Renewal and Regeneration.

The hematopoietic system is highly organized to maintain its functional integrity and to meet lifelong organismal demands. Hematopoietic stem cells (HSCs) must balance self-renewal with differentiation and the regeneration of the blood system. It is a complex balancing act between these competing HSC functions. Although highly quiescent at steady state, HSCs become activated in response to inflammatory cytokines and regenerative challenges. This activation phase leads to many intrinsic stresses such as replicative, metabolic, and oxidative stress, which can cause functional decline, impaired self-renewal, and exhaustion of HSCs. To cope with these insults, HSCs use both built-in and emergency-triggered stress-response mechanisms to maintain homeostasis and to defend against disease development. In this review, we discuss how the hematopoietic system operates in steady state and stress conditions, what strategies are used to maintain functional integrity, and how deregulation in the balance between self-renewal and regeneration can drive malignant transformation.

Medicinal Uses of Hematopoietic Growth Factors in Neonatal Medicine.

This review focuses on certain hematopoietic growth factors that are used as medications in clinical neonatology. It is important to note at the chapter onset that although all of the pharmacological agents mentioned in this review have been approved by the US Food and Drug administration for use in humans, none have been granted a specific FDA indication for neonates. Thus, in a sense, all of the agents mentioned in this chapter could be considered experimental, when used in neonates. However, a great many of the pharmacological agents utilized routinely in neonatology practice do not have a specific FDA indication for this population of patients. Consequently, many of the agents reviewed in this chapter are considered by some practitioners to be nonexperimental and are used when they judge such use to be "best practice" for the disorders under treatment.The medicinal uses of the agents in this chapter vary considerably, between geographic locations, and sometimes even within an institutions. "Consistent approaches" aimed at using these agents in uniform ways in the practice of neonatology are encouraged. Indeed some healthcare systems, and some individual NICUs, have developed written guidelines for using these agents within the practice group. Some such guidelines are provided in this review. It should be noted that these guidelines, or "consistent approaches," must be viewed as dynamic and changing, requiring adjustment and refinement as additional evidence accrues.

[Modalities of mobilization and hematopoietic stem cells objectives in autologous transplantation: Guidelines from the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)]. / Modalités de mobilisation des cellules souches hématopoïétiques autologues et objectifs cellulaires en cellules CD34+ : recommandations de la Société francophone de greffe de mœlle et de thérapie cellulaire (SFGM-TC).

The modalities of mobilization of hematopoietic stem cells in autologous transplantation have evolved in recent years. The Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC) organized the 9th hematopoietic stem cell transplantation clinical practices harmonization workshop series in September 2018 in Lille, France, to conduct a review of current practices of the society centers and of international recommendations. The cell dose objectives have been revised. The modalities of mobilization including the use of plerixafor have been specified allowing reaching the objectives of collection while limiting the number of apheresis. Collections failures have become exceptional.

Protective effects of Clec11a in islets against lipotoxicity via modulation of proliferation and lipid metabolism in mice.

The lipotoxicity is considered as one of the risk for diabetes. Here we report C-type lectin domain family 11, member A (Clec11a) as a new regulator in islet playing a protective role in lipotoxicity induced dysfunction. Islet transcriptome sequencing was performed using the high-fat diet induced obesity (DIO) mice model. We found a significant decrease of Clec11a expression in islets of DIO mice compared to normal control mice, which was further confirmed by real-time PCR. Immunostaining demonstrated the localization of the Clec11a protein in mouse islets. Administration of recombinant human Clec11a (rClec11a) protein promoted the proliferation of islet cells and rescued the inhibition of fatty acid on cell proliferation, which involved the activation of Erk signaling pathway. We also found that the rClec11a altered the expression of genes involved in lipid metabolism.

Targeted genetic screening in mice through haploid embryonic stem cells identifies critical genes in bone development.

Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- mice exhibited small skeletal size only at birth, Irx5-/- mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.