Effects of Stem Cell Factor/c-Kit Signaling on In Vitro Maturation of Porcine Oocytes and Subsequent Developmental Competence After Fertilization

Stem cell factor (SCF), also known as c-Kit ligand, plays an important role in the proliferation of primordial germ cells and the survival of oocytes during follicular development. The aim of this study was to investigate the effect of SCF/c-Kit signaling on in vitro maturation (IVM) of porcine oocytes by analyzing nuclear and cytoplasmic maturation, oocyte size, cumulus cell expansion, and developmental competence to the blastocyst stage. Moreover, mRNA expression patterns of porcine cumulus cells and oocytes were evaluated using qRT-PCR. Following 42 h of IVM, 10 and 50 ng/mL SCF-treated groups exhibited significantly (P < 0.05) increased polar body extrusion rates and intracellular glutathione levels compared with the control group. The cumulus expansion index significantly (P < 0.05) increased in all SCF-treated groups compared with the control samples. mRNA levels of the proapoptotic gene Bax and apoptosis-related cysteine peptidase Caspase3 were lower in SCF-treated cumulus cells than in the control group. Notably, the diameter of oocytes after IVM, the mRNA expression of well-known oocyte-secreted factors (GDF9 and BMP15), and an oocyte-specific protein essential for ovulation and oocyte health (YBX2) were significantly (P < 0.05) higher in SCF-treated than in non-treated oocytes. Inhibition of c-Kit during porcine IVM using ACK2, an antagonistic blocker of c-Kit, significantly (P < 0.05) decreased the polar body extrusion rate compared with the control, as well as blastocyst formation rate compared with the 10 ng/mL SCF-treated group. In conclusion, the effect of SCF/c-Kit-mediated signaling during porcine IVM could be ascribed to the reduced expression of apoptosis-related genes and higher expression of oocyte-specific/secreted factors.

Hematopoietic Recovery using Multi-Cytokine Therapy in 8 Patients Presenting Radiation-Induced Myelosuppression after Radiological Accidents

Treatment of accidental radiation-induced myelosuppression is primarily based on supportive care and requires specific treatment based on hematopoietic growth factors injection or hematopoietic cell transplantation for the most severe cases. The cytokines used consisted of pegylated erythropoietin (darbepoetin alfa) 500 IU once per week, pegylated G-CSF (pegfilgrastim) 6 mg × 2 once, stem cell factor 20 μg.kg–1 for five days, and romiplostim (TPO analog) 10 μg.kg−1 once per week, with different combinations depending on the accidents. As the stem cell factor did not have regulatory approval for clinical use in France, the French regulatory authorities (ANSM, formerly, AFSSAPS) approved their compassionate use as an investigational drug “on a case-by-case basis”. According to the evolution and clinical characteristics, each patient's treatment was adopted on an individual basis. Daily blood count allows initiating G-CSF and SCF delivery when granulocyte <1,000/mm3, TPO delivery when platelets <50,000/mm3, and EPO when Hb<80 g/L. The length of each treatment was based on blood cell recovery criteria. The concept of “stimulation strategy” is linked to each patient's residual hematopoiesis, which varies among them, depending on the radiation exposure's characteristics and heterogeneity. This paper reports the medical management of 8 overexposed patients to ionizing radiation. The recovery of bone marrow function after myelosuppression was accelerated using growth factors, optimized by multiple-line combinations. Particularly in the event of prolonged exposure to ionizing radiation in dose ranges inducing severe myelosuppression (in the order of 5 to 8 Gy), with no indication of hematopoietic stem cell transplantation.

A polarized anchor for hematopoietic stem cells: Synapse between stem cells and their niche?

Multipotent hematopoietic stem cells are maintained by the bone marrow niche, but how niche-derived membrane-bound stem cell factor (mSCF) regulates HSCs remains unclear. In this issue, Hao et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202010118) describe that mSCF, synergistically with VCAM-1, induces large, polarized protrusions that serve as anchors for HSCs to their niche.

LncRNA TCL6 Contributes to CSC-like Properties via Modulating TP53 in HCC

BackgroundHepatocellular carcinoma (HCC) is the most common malignant tumors, accounting for most of the adult primary liver cancer. Herein, we aimed to analyze the expression of long non-coding RNA-T cell leukemia/lymphoma 6 (lncRNA-TCL6) in HCC and elucidate its mechanism involved in the HCC progression. Methodse performed RNA extraction and quantitative real-time polymerase chain reaction assays, spheroid formation assays, flow cytometry and western blot assays to assess the effect of TCL6 on the liver CSCs marker CD133 expression rate, sphere-forming ability of liver stem cells, and the relationship between TCL6 expression and stem cell factor (TP53, P21, CD44, KLF4, OCT4, Nanog, and Sox2). In addition, we used a dual luciferase assay to verify the relationship between miR-106a-5p and TP53.ResultsKnockdown of TCL6 expression significantly improved the CD133 expression rate and the liver stem cells sphere-forming ability in HCC, while TCL6 overexpression in HCC showed the opposite effect. Knockdown of TCL6 upregulated the KLF4mRNA expression, while TCL6 overexpression in HCC inhibited the TP53 and CDKN1A expression. Western blot assays showed that TCL6 expression was positively correlated with TP53 and P21, while negatively correlated with stem cell factor. Dual luciferase assay showed that TP53 was a target of miR-106a-5p.ConclusionResults suggested that reprogramming-related TCL6 may be a novel tumor suppressor gene in HCC, which inhibits the self-renewal of liver CSCs, in part by promoting the TP53 expression.

Microbiota-derived lactate promotes hematopoiesis and erythropoiesis by inducing stem cell factor production from leptin receptor+ niche cells

Although functional interplay between intestinal microbiota and distant sites beyond the gut has been identified, the influence of microbiota-derived metabolites on hematopoietic stem cells (HSCs) remains unclear. This study investigated the role of microbiota-derived lactate in hematopoiesis using mice deficient in G-protein-coupled receptor (Gpr) 81 (Gpr81−/−), an established lactate receptor. We detected significant depletion of total HSCs in the bone marrow (BM) of Gpr81−/− mice compared with heterogenic (Gpr81+/−) mice in a steady state. Notably, the expression levels of stem cell factor (SCF), which is required for the proliferation of HSCs, decreased significantly in leptin receptor-expressing (LepR+) mesenchymal stromal cells (MSCs) around the sinusoidal vessels of the BM from Gpr81−/− mice compared with Gpr81+/− mice. Hematopoietic recovery and activation of BM niche cells after irradiation or busulfan treatment also required Gpr81 signals. Oral administration of lactic acid-producing bacteria (LAB) activated SCF secretion from LepR+ BM MSCs and subsequently accelerated hematopoiesis and erythropoiesis. Most importantly, LAB feeding accelerated the self-renewal of HSCs in germ-free mice. These results suggest that microbiota-derived lactate stimulates SCF secretion by LepR+ BM MSCs and subsequently activates hematopoiesis and erythropoiesis in a Gpr81-dependent manner.

Stimulation of c-Kit+ Retinal Progenitor Cells by Stem Cell Factor Confers Protection Against Retinal Degeneration

Background c-Kit/CD117, expressed in a series of tissue-specific progenitor cells, plays an important role in tissue regeneration and tissue homeostasis. We previously demonstrated that organoid-derived c-Kit+ retinal progenitor cells can facilitate the restoration of degenerated retina. Meanwhile, we have identified a population of endogenous c-Kit+ cells in retinas of adult mouse. However, the exact role of these cells in retinal degeneration remains unclear. Methods Retinal degeneration was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Two days post NMDA challenge, intravitreal injection of stem cell factor (SCF) was performed. Distribution and abundance of c-Kit+ cells and other retinal cells were evaluated by immunochemistry. Retinal function of treated mice was tested via flash electroretinogram (fERG) and the light/dark transition test. Possible regulatory pathways were evaluated by RNA sequencing. Results NMDA challenge increased the total number of c-Kit+ cells in the retinal ganglion cell layer (GCL), while slightly deregulated the protein level of SCF, which is mainly expressed in Müller cells. Both fERG and light/dark transition tests showed that intravitreal injection of SCF effectively improve the visual function of NMDA-treated mice. Consistently, the activation of microglia in injured retina has also been inhibited after SCF treatment. Mechanistically, SCF administration not only prevent the loss of retinal ganglion cells (RGCs), but also maintained the function of RGCs as quantified by fERG. Further, we performed transcriptome sequencing analysis of the retinal cells isolated from SCF-treated mice and the parallel control. Gene Ontology analysis showed that SCF-induced transcriptome changes were closely correlated with eye development-related pathways. Crystallins and several protective factors such as Pitx3 were significantly upregulated by SCF treatment. Conclusions Our results revealed the role of c-Kit+ cells in the protection of RGCs in NMDA-treated mice, via inhibiting the loss of RGCs. Administration of SCF can act as a potent strategy for treating retinal degeneration-related diseases.

Chromatin lncRNA Platr10 controls stem cell pluripotency by coordinating an intrachromosomal regulatory network

Background A specific 3-dimensional intrachromosomal architecture of core stem cell factor genes is required to reprogram a somatic cell into pluripotency. As little is known about the epigenetic readers that orchestrate this architectural remodeling, we used a novel chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to profile long noncoding RNAs (lncRNAs) in the Oct4 promoter. Results We identify Platr10 as an Oct4 - Sox2 binding lncRNA that is activated in somatic cell reprogramming. Platr10 is essential for the maintenance of pluripotency, and lack of this lncRNA causes stem cells to exit from pluripotency. In fibroblasts, ectopically expressed Platr10 functions in trans to activate core stem cell factor genes and enhance pluripotent reprogramming. Using RNA reverse transcription-associated trap sequencing (RAT-seq), we show that Platr10 interacts with multiple pluripotency-associated genes, including Oct4, Sox2, Klf4, and c-Myc, which have been extensively used to reprogram somatic cells. Mechanistically, we demonstrate that Platr10 helps orchestrate intrachromosomal promoter-enhancer looping and recruits TET1, the enzyme that actively induces DNA demethylation for the initiation of pluripotency. We further show that Platr10 contains an Oct4 binding element that interacts with the Oct4 promoter and a TET1-binding element that recruits TET1. Mutation of either of these two elements abolishes Platr10 activity. Conclusion These data suggest that Platr10 functions as a novel chromatin RNA molecule to control pluripotency in trans by modulating chromatin architecture and regulating DNA methylation in the core stem cell factor network.