Differentiation Of Progenitor Stem Cells Research Articles

Cancer Moonshot: Moonshot as a Magic Code to Guide Successful Solutions of Tough Challenges Such as Cancer

Presidential projects deal with monumentally important issues. Moonshot and war on cancer were two unrelated presidential projects: the moonshot project was successful, but the war on cancer project was unsuccessful. President Biden brought up these two presidential projects on Sept. 12, 2022 apparently with an intention to urge the health profession to learn from the success of moonshot to come up solutions to save 50% of cancer patients in the following 25 years. The goal of cancer moonshot was modest. The health profession, however, must make a drastic change on the approach of cancer therapy to fulfill cancer moonshot. Moonshot was an extremely difficult challenge. So far, the USA was the only nation able to achieve this difficult challenge. Evidently, the right approach to a difficult challenge was the magic code to the success of moonshot. On the other hand, a wrong approach to a simple matter might result in making simple problem unsolvable. Apparently, war on cancer was such a case. Cytotoxic chemotherapy and radiotherapy based on the killing of cancer cells were the choice of cancer establishments to wage the war on cancer. The approach was wrong, because cancer was a disease due to wound unhealing. Creating more wounds definitely was a wrong approach. The war on cancer was loomed to fail on the day of its declaration. The wrong approach continued to dominate cancer therapies with no hope in sight to successfully saving cancer patients. Cancer moonshot was in essence a protest of the failure to save cancer patients from the highest government official to the health profession. A change of approach on cancer therapy is obviously needed to save cancer patients. Since cancer is caused by wound unhealing. Wound healing process is the most appropriate modality of cancer therapy. Wound healing comes naturally, because the nature creates chemo-surveillance to ensure perfection of wound healing. Wound healing requires the proliferation and the terminal differentiation of progenitor stem cells (PSCs). The success of wound healing depends on the efficient differentiation of PSCs to eliminate the symptom created by the wound. Chemo-surveillance plays such an important role to dictate the success of wound healing. Chemo-surveillance can be damaged under pathological conditions to produce tumor necrosis factor (TNF) to cause cachexia symptoms. Inability of PSCs to undergo terminal differentiation due to the collapse of chemo-surveillance then forces PSCs to evolve into cancer stem cells (CSCs) and then to progress to faster growing cancer cells (CCs). Obviously, cancer arises due to the collapse of chemo-surveillance. Restoration of chemo-surveillance with CDA formulations is, therefore, the right approach of cancer therapy. The wisdom of the nature beats the wisdom of cancer establishments on the issue of cancer.

Wound Healing Process as the Most Appropriate Modality of Cancer Therapy

President Biden of USA declared cancer moonshot initiative on Sept. 12, 2022 to urge the health profession to come up solutions to save 50% of cancer patients in the following 25 years. Cancer therapies were dominated by strategy based on killing of cancer cells in the past, which have been drilled through as a presidential project, but failed to achieve the goal to win the war on cancer. Obviously, a drastic change of strategy is necessary to fulfill moonshot of President Biden. Wound healing requires the proliferation and the terminal differentiation of progenitor stem cells (PSCs). Wound healing comes naturally, because the nature creates chemo-surveillance to ensure perfection of wound healing. Healthy people are able to maintain a steady level of wound heading metabolites active as differentiation inducers (DIs) and differentiation helper inducers (DHIs) to promote terminal differentiation of PSCs through destabilization of abnormal methylation enzymes (MEs). The functionality of chemo-surveillance can be damaged under pathological conditions triggering the production of tumor necrotic factor (TNF) to cause cachexia symptoms. A manifestation of cachexia symptoms is the excessive urinary excretion of wound healing metabolites to affect terminal differentiation of PSCs to heal wound, allowing PSCs to evolve into cancer stem cells (CSCs), and then to progress to faster growing cancer cells (CCs) through activation of oncogenes and/or inactivation of suppressor genes. Since cancer is caused by wound unhealing. Thus, wound healing process offers the most appropriate modality of cancer therapy. CDA-2 is a preparation of wound healing metabolites purified from urine, which has superb therapeutic efficacy against myelodysplastic syndrome (MDS). MDS is a disease caused by the build up of CSCs unable to undergo terminal differentiation. Apparently, wound healing metabolites are the best medicine to eradicate CSCs. For the therapy of cancer, we propose to add another set of DI + DHI consisting of synthetic products to target CCs, since CCs are known to express high levels of salvage enzymes to support their faster growth. CDA formulations definitely can fulfill cancer moonshot. Perpetual replication of CCs is the most outstanding feature of cancer. Naturally, killing of CCs becomes the choice of cancer establishments to combat cancer in the past. This modality of cancer therapy has been drilled through as a presidential project, but failed to achieve the goal to win the war on cancer. The contribution to damage chemo-surveillance and the inability to eradicate CSCs are responsible for the failure to save cancer patients. A combination therapy with CDA formulations can eliminate such deficit to fulfill cancer moonshot.

Cell Differentiation Agent Formulations to Win the War on Cancer

Solution of cancer was obviously an important national interest for President Nixon to declare ‘War on Cancer” in 1971. Health professionals failed the challenge to put cancer away during the 5 years of intensive presidential support and in the following 46 years of exclusive medical support allocated to cancer. Cancer stem cells (CSCs) stood in the way to deny the success of cytotoxic agents, the choice of cancer establishments in the past, to put cancer away. Cancer arises due to wound not healing properly. Consequently, the process of wound healing is the most appropriate modality of cancer therapy. Cytotoxic agents creating wounds are apparently contra-indication on cancer therapy. Cytotoxic agents are, however, the most effective agents to eliminate the most outstanding feature of cancer, namely the perpetual replication of cancer cells (CCs). A critical mechanism of wound healing is the induction of terminal differentiation of progenitor stem cells (PSCs) which are the precursors of CSCs. Wound healing metabolites active as differentiation inducers (DIs) and differentiation helper inducers (DHIs) are required to direct the terminal differentiation of PSCs to heal the wound. Cell differentiation agent (CDA) formulations are preparations of wound healing metabolites most fit to take out PSCs and CSCs by directing these cells to undergo terminal differentiation, which are protected by drug resistance and anti-apoptosis mechanisms, thus unresponsive to cytotoxic agents. CDAs alone or in combination with cytotoxic agents become perfect drugs to take out both CCs and CSCs, and to restore the functionality of chemo-surveillance necessary to win the war on cancer.

The Induction of Bone Formation by the recombinant human transforming growth Factor-β3 : From preclinical studies in Papio ursinus to translational research in Homo sapiens

Skeletal bone defects of the axial or the craniomaxillofacial skeletons still present formidable challenges to skeletal reconstructionists, tissue biologists and modern medicine. In systematic research experiments in the Chacma baboon Papio ursinus our laboratories have shown the previously unreported osteoinductive activity of the three mammalian transforming growth factor-β (TGF-β) isoforms. This review discusses the induction of bone formation by the mammalian TGF-βs with particular reference to the substantial and rapid induction of bone by the recombinant hTGF-β3 from the laboratory benches, to pre-clinical studies in heterotopic and orthotopic mandibular sites of Papio ursinus to clinical translation in human patients. Design and MethodsA series of systematic research experiments in Papio ursinus using the hTGF-β3 together with earlier experiments using the -β1 and β2 isoforms are reviewed and re-analyzed molecularly nd morphologically to provide the basic research data for the reported clinical translation in human patients. Results The three mammalian hTGF-β isoforms and notably hTGF-β3 induce rapid and substantial induction of heterotopic bone in intramuscular sites of Papio ursinus. Relatively low doses of hTGF-β1or hTGF-β3 in binary application with hBMP-7 synergize to induce massive corticalized ossicles in the rectus abdominis muscle. In orthotopic mandibular sites, 125 and250 µg doses of hTGF-β3 induce bone formation across large mandibular defects in Papio ursinus with corticalized buccal and lingual plates by day 30, with modeling and maintenance of corticalized bone by 9 to 12 months after implantation of the 250 µg dose in 3 cm mandibular defects Papio ursinus. Discussion hTGF-β3 significantly up-regulates RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone by the hTGF-β3 is via the bone morphogenetic proteins pathway; hTGF-β3 controls theinduction of bone by regulating the expression of BMPs gene and gene products via Noggin expression, eliciting bone induction by up-regulating exogenous BMPs.

No Scar as an Indication of Perfect Wound Healing, Ugly Scar as Imperfect Wound Healing and Cancer as Failed Wound Healing

Wound healing and cancer evolution are closely related to involve progenitor Stem Cells (PSCs) as the critical common elements. Wound healing requires the proliferation and the Terminal Differentiation (TD) of PSCs. Wound triggers biological and immunological responses. The biological response involves the release of Arachidonic acId (AA) from membrane bound phosphatidylinositol for the synthesis of prostaglandins (PGs) which are active differentiation inducers (DIs) good for wound healing. Immunological response prompts the production of Tumor Necrosis Factor (TNF), which is also named cachectin after its effect to cause cachexia symptom. Cachexia symptom is bad for wound healing, because of excessive urinary excretion of low molecular weight metabolites resulting in the loss of wound healing metabolites. The proliferation of PSCs is promoted by PGs and the TD of PSCs is accomplished by metabolites involved in chemo-surveillance. Thus, the functionality of chemo-surveillance plays an important role to dictate the success of wound healing. If the functionality is perfect as healthy people, healing of wound with no scar can always be expected. But if the functionality of chemo-surveillance has been damaged due to pathological conditions causing cachexia symptom, then healing of wound may be impaired to result in ugly scar, or even worse cancer. Ugly scar in visible surface is a medical concern, particularly with respect to cosmetic surgery. Cancer is a very fearful disease. The study of wound healing is helpful to heal wound with no scar and to search for a more appropriate strategy of cancer therapy. Data in support of this opinion article were produced by Ming C. Liau as a volunteer researcher during 2010 to 2020 in the laboratory of Professor John P Fruehauf at Chao Family Comprehensive Cancer Center, University of California Irvine Medical Center, CA,USA.

The Role of Congenital Cytomegalovirus Infection in Adverse Birth Outcomes: A Review of the Potential Mechanisms.

Human cytomegalovirus (CMV) is a major cause of nonhereditary adverse birth outcomes, including hearing and visual loss, neurologic deficits, and intrauterine growth retardation (IUGR), and may contribute to outcomes such as stillbirth and preterm delivery. However, the mechanisms by which CMV could cause adverse birth outcomes are not fully understood. This study reviewed proposed mechanisms underlying the role of CMV in stillbirth, preterm birth, and IUGR. Targeted literature searches were performed in PubMed and Embase to identify relevant articles. Several potential mechanisms were identified from in vitro studies in which laboratory-adapted and low-passage strains of CMV and various human placental models were used. Potential mechanisms identified included impairment of trophoblast progenitor stem cell differentiation and function, impairment of extravillous trophoblast invasiveness, dysregulation of Wnt signaling pathways in cytotrophoblasts, tumor necrosis factor-α mediated apoptosis of trophoblasts, CMV-induced cytokine changes in the placenta, inhibition of indoleamine 2,3-dioxygenase activity, and downregulation of trophoblast class I major histocompatibility complex molecules. Inherent challenges for the field remain in the identification of suitable in vivo animal models. Nonetheless, we believe that our review provides useful insights into the mechanisms by which CMV impairs placental development and function and how these changes could result in adverse birth outcomes.

P63 and Its Target Follistatin Maintain Salivary Gland Stem/Progenitor Cell Function through TGF-β/Activin Signaling.

SummaryMultipotent ΔNp63-positive cells maintain all epithelial cell lineages of the embryonic and adult salivary gland (SG). However, the molecular mechanisms by which ΔNp63 regulates stem/progenitor (SP) cell populations in the SG remains elusive. To understand the role of ΔNp63 in directing cell fate choices in this gland, we have generated ΔNp63-deleted adult mice and primary salivary cell cultures to probe alterations in SP cell differentiation and function. In parallel, we have leveraged RNA-seq and ChIP-seq-based characterization of the ΔNp63-driven cistrome and scRNA-seq analysis to molecularly interrogate altered SG cellular identities and differentiation states dependent on ΔNp63. Our studies reveal that ablation of ΔNp63 results in a loss of the SP cell population and skewed differentiation that is mediated by Follistatin-dependent dysregulated TGF-β/Activin signaling. These findings offer new revelations into the SP cell gene regulatory networks that are likely to be relevant for normal or diseased SG states.

HIV-1 inhibits haematopoiesis via microRNA secreted by virus-infected CD4+ T cells.

HIV-1-infected patients develop haematological disorders such as cytopenias. One possible explanation is the inhibition of haematopoiesis at the level of differentiation of CD34+ haematopoietic progenitor stem cells. Based on our previous studies, we hypothesised that there may be viral encoded, or host cellular factors which participate in the process of inhibition of haematopoiesis. Virus-depleted media from infected CD4+ T cells was prepared by filtration and added to CD34+ cell differentiation semisolid medium. We have also used the virus-depleted media to isolate host/viral factors including miRNA. Isolated miRNAs were screened for their haematopoietic inhibitory function using the miRNA mining approach. Addition of virus-depleted media caused a 40% inhibition of differentiation of CD34+ cells into myeloid and erythroid colony formation. Real-time RT-PCR showed miR-15a and miR-24 from both pIndie-C1 and pNL4.3 HIV-1-infected cells showed a significant differential expression when compared to control media. In this study, we have identified two miRNAs, miR-15a and miR-24 secreted from purified HIV-1-infected CD4+ T cells that inhibited CD34+ haematopoietic progenitor stem cell differentiation into myeloid and erythroid colonies in vitro.

Clodronate as a Therapeutic Strategy against Osteoarthritis.

Osteoarthritis (OA), the most prevalent musculoskeletal pathology, is mainly characterized by the progressive degradation of articular cartilage due to an imbalance between anabolic and catabolic processes. Consequently, OA has been associated with defects in the chondrocitic differentiation of progenitor stem cells (PSCs). In addition, SOX9 is the transcription factor responsible for PSCs chondrogenic commitment. To evaluate the effects of the non-amino bisphosphonate clodronate in OA patients we investigated SOX9 gene expression in circulating progenitor cells (CPCs) and in an in vitro OA model. We evaluated pain intensity, mental and physical performance in OA patients, as well as serum biomarkers related to bone metabolism. In addition, in order to improve therapeutic strategies, we assayed nanoparticle-embedded clodronate (NPs-clo) in an in vitro model of chondrogenic differentiation. Our data showed upregulation of SOX9 gene expression upon treatment, suggesting an increase in chondrocytic commitment. Clodronate also reduced osteoarticular pain and improved mental and physical performance in patients. Furthermore, NPs-clo stimulated SOX9 expression more efficaciously than clodronate alone. Clodronate may therefore be considered a good therapeutic tool against OA; its formulation in nanoparticles may represent a promising challenge to counteract cartilage degeneration.

In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained After Mechanical Digestion of Human Dental Pulp.

Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio-maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. J. Cell. Physiol. 232: 548-555, 2017. © 2016 Wiley Periodicals, Inc.

Secretome profiling of cytokines and growth factors reveals that neuro-glial differentiation is associated with the down-regulation of Chemokine Ligand 2 (MCP-1/CCL2) in amniotic fluid derived-mesenchymal progenitor cells.

Secreted cytokines and growth factors play a key role in the modulation of stem cell proliferation, differentiation and survival. To investigate the interplay between the changes in their expression levels, we used the newly characterized human amniotic fluid derived-mesenchymal progenitor MePR-2B cell line differentiated to a neuro-glial phenotype and exploited the very high sensitivity and versatility of magnetic beads-based immunoassays. We found that a sub-set of proteins, including the cytokines IL-6, TNFα, IL-15, IFNγ, IL-8, IL-1ra, MCP-1/CCL2, RANTES and the growth factor PDGFbb, underwent a significant down-regulation following neuro-glial differentiation, whereas the expression levels of IL-12 p70, IL-5, IL-7, bFGF, VEGF and G-CSF were increased. The role of MCP-1/CCL2, previously identified as a regulator of neural progenitor stem cell differentiation, has been further investigated at transcriptional level, revealing that both the chemokine and its receptor are co-expressed in MePR-2B cells and that are regulated upon differentiation, suggesting the presence of an autocrine and paracrine loop in differentiating cells. Moreover, we demonstrated that exogenous CCL2 is capable to affect neuro-glial differentiation in MePR-2B cells, thus providing novel evidences for the potential involvement of chemokine-mediated signaling in progenitor/stem cells differentiation processes and fate specification.

Bone-like mineral nucleating peptide nanofibers induce differentiation of human mesenchymal stem cells into mature osteoblasts.

A bone implant should integrate to the tissue through a bone-like mineralized interface, which requires increased osteoblast activity at the implant-tissue boundary. Modification of the implant surface with synthetic bioinstructive cues facilitates on-site differentiation of progenitor stem cells to functional mature osteoblasts and results in subsequent mineralization. Inspired by the bioactive domains of the bone extracellular matrix proteins and the mussel adhesive proteins, we synthesized peptide nanofibers to promote bone-like mineralization on the implant surface. Nanofibers functionalized with osteoinductive collagen I derived Asp-Gly-Glu-Ala (DGEA) peptide sequence provide an advantage in initial adhesion, spreading, and early commitment to osteogenic differentiation for mesenchymal stem cells (hMSCs). In this study, we demonstrated that this early osteogenic commitment, however, does not necessarily guarantee a priority for maturation into functional osteoblasts. Similar to natural biological cascades, early commitment should be further supported with additional signals to provide a long-term effect on differentiation. Here, we showed that peptide nanofibers functionalized with Glu-Glu-Glu (EEE) sequence enhanced mineralization abilities due to osteoinductive properties for late-stage differentiation of hMSCs. Mussel-inspired functionalization not only enables robust immobilization on metal surfaces, but also improves bone-like mineralization under physiologically simulated conditions. The multifunctional osteoinductive peptide nanofiber biointerfaces presented here facilitate osseointegration for long-term clinical stability.

The induction of bone formation by the recombinant human transforming growth factor-β3

Implantation of recombinant human transforming growth factor-β3 (hTGF-β3) with coral-derived calcium carbonate-based macroporous bioreactors with limited conversion to hydroxyapatite (7% HA/CC) in the rectus abdominis muscle of the non-human primate Chacma baboon Papio ursinus induces endochondral bone formation. The exact mechanisms by which hTGF-β3 signalling induces bone in heterotopic sites of P. ursinus are not known. Coral-derived 7% HA/CC bioreactors with and without 125 μg hTGF-β3 were implanted in triplicate in the rectus abdominis muscle of 6 adult baboons. 7% HA/CC bioreactors either with or without hTGF-β3 were loaded with 125 μg of recombinant human Noggin (hNoggin), a bone morphogenetic proteins (BMPs) antagonist. Tissues on day 15, 60 and 90 were analysed by histomorphometry and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Down-regulation of BMP-2 characterized 7% HA/CC constructs preloaded with 125 μg hNoggin with Noggin down-regulated on day 60 and 90 together with lack of TGF-β3 expression. Down-regulation of BMP-2 correlated with minimal bone formation by induction. hTGF-β3/hNoggin pre-treated bioreactors up-regulated BMP-2 but only on day 90 together with a significant down-regulation of Noggin on day 60 and 90, correlating with the induction of bone formation, albeit limited, on day 90 at the periphery of the macroporous bioreactors only. hTGF-β3 treated bioreactors significantly down-regulated BMP-2 on day 15 whilst up-regulating BMP-2 on day 60 and 90, together with down-regulation of Noggin on day 60 and 90 correlating with the prominent induction of bone formation. hTGF-β3 significantly up-regulated RUNX-2 and Osteocalcin expression on day 15 controlling the differentiation of progenitor stem cells into the osteoblastic lineage. The induction of bone as initiated by hTGF-β3 in the rectus abdominis muscle of P. ursinus is via the BMPs pathway with hTGF-β3 controlling the induction of bone formation by regulating the expression of BMPs via Noggin expression. These results unequivocally demonstrate that hTGF-β3 elicits bone induction by up-regulation of endogenous BMP-2 and is blocked by hNoggin.

Thrombocytopenia in HIV Infection: Impairment of Platelet Formation and Loss Correlates with Increased c-Mpl and Ligand Thrombopoietin Expression

Thrombocytopenia is a common hematologic disorder in patients infected with the human immunodeficiency virus (HIV) and represents a risk for bleeding which is further deleterious during surgery. The major causes of the thrombocytopenia include accelerated peripheral platelet destruction by antiplatelet antibodies and insufficient production of platelets from the infected megakaryocytes. Additionally, at an earlier stage of platelet development, HIV may inhibit megakaryopoiesis at multiple stages of pluripotent CD34+ progenitor stem cell differentiation possibly contributing to decreased levels of platelets in circulation. In HIV infected patients, both the serum thrombopoietin (TPO) levels and theTPO-c-Mpl complexes on the platelet surface were significantly elevated. Therapeutic infusion of HIV infected patients with pegylated recombinant human megakaryocyte growth development factor (PEG-rHu-MGDF) restores platelet counts to normal levels and reduces the c-Mpl expression per platelet. In vitro aggregation of platelets treated with TPO and agonist, adenosine diphosphate (ADP), decrease the dose of ADP that is required for half-maximum aggregation. In vivo dosing does not effect platelet aggregation showing that the metabolism of TPO following its internalization through TPO-c-Mpl complex is rapid and that dosing within the therapeutic range does not constitute increased risk of thrombotic disease.

Bio-Inspired Morphogens and Biomimetic Biomaterials for Regenerative Medicine

AbstractRegenerative medicine is the science of design and manufacture of parts for functional restoration of damaged tissues due to cancer, disease and trauma. Morphogenesis is the developmental cascade of pattern formation, body plan establishment and differentiation of tissues culminating in adult form. Regeneration in general, recapitulates in parts, embryonic morphogenesis. Thus, the principles of morphogenesis can be applied to tissue engineering for regenerative medicine and surgery. The threekey ingredients for tissue engineering and regenerative medicine are inductive morphogens, responding stem cells, and extracellular matrix materials. Therefore bioactive morphogens can be integrated into materials for functional restoration by tissue engineering. A morphogen is a morphogenetic protein signal that acts on responding stem cells. Bone morphogenesis is induced by bone morphogenetic proteins (BMPs). BMPs play a role in pattern formation, cell differentiation, maintenance and regeneration of tissues. BMPs are pleiotropic and act on chemotaxis, mitosis and differentiation of progenitor stem cells. There are nearly twenty BMPs in the human genome. BMPs have actions beyond bone in development of teeth, heart, kidney, eye, skin, and brain. Thus, BMPs may be called body morphogenetic proteins. Stem cells are primordial cells with unlimited replicative potential and can be programmed by morphogens such as BMPs. Extracellular matrix is the native scaffolding material that can be used to deliver morphogens such as BMPs for tissue engineering of bone. Biomimetic materials are typically synthetic, polymeric structures that mimic extracellular matrix scaffolding by presenting the cellular binding sites found within collagens, fibronectin, proteoglycans and glycoproteins. Materials that mimic extracellular matrix scaffolding such as BMPs bind to collagens I&IV, heparan sulfate and heparin. BMPs bound to collagen acts as a composite biomaterial to initiate bone formation and the shape can be molded by an appropriate template. In addition to including BMPs in a carrier matrix, the geometry of the carrier matrix is critical for optimal tissue engineering. For example: collagen particles smaller than 44μm are feeble in bone induction compared to the coarse (420μm) particles. Gene therapy approaches using genes for morphogenesis such as BMPs allows a sustained, prolonged secretion of gene products. Thus, morphogens integrated into biomaterials may be useful in regenerative medicine.

Regulation by pH of the Alternative Splicing of the Stem Cell Factor Pre-mRNA in the Testis

Proliferation and differentiation of progenitor stem cells are mainly controlled by diffusible and adhesion molecules. Stem cell factor (SCF), an essential regulator of spermatogenesis produced by Sertoli cells, utilize both modes of cell to cell communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound (SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that the flanking intronic sequences perfectly follow the gt-at rule, suggesting that the basal splicing machinery might not be responsible by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells mainly express SCFm, but a switch to SCFs occurs after 48 h of culture. We found that this change can be prevented by acidification of the culture medium at pH 6.3 or by addition of lactate. The sustained synthesis of SCFm at low pH was no longer observed in the presence of cycloheximide, suggesting that SCF exon 6 skipping requires de novo protein synthesis. Accordingly, UV cross-linking experiments show that nuclear Sertoli cell protein(s) bind in a sequence-specific manner to exon 6. Together, our data allow the proposal of an integrated mechanism in which the synthesis of lactate by Sertoli cells is used in the same time as an energetic substrate for germ cells and as a promoter of their survival/proliferation through the production of SCFm.