Potent Inducer Of Differentiation Research Articles

Preparation of kartogenin-loaded PLGA microspheres and a study of their drug release profiles

Introduction: Kartogenin, a potent inducer of chondrogenic differentiation in mesenchymal stem cells and a key agent in cartilage regeneration, presents a viable therapeutic strategy for osteoarthritis management. Despite the abundance of literature on therapeutic potential of kartogenin, there is a paucity of studies characterizing the formulation specifics in microsphere fabrication. This exploration is pivotal to advances in regenerative medicine, particularly in the domain of cartilage regeneration, to assure clinical efficacy and safety.Methods: In this work, we fabricated kartogenin-loaded PLGA microspheres with diverse formulations and their particle size, size distribution, encapsulation efficiency, drug loading and release profiles were characterized. Ratio of polymer, drug, and solvent and the use of surfactant was used as variables, and in particular, the effect of surfactant on particles was investigated.Results: The average diameter of the spheres was 16.0–31.7 μm. Morphological variations from solid to porous surface structures depending on surfactant incorporation during the emulsification process was observed. Cumulative kartogenin release from microspheres ranged from 53.8% to 80.9% on day 28, and release profiles conform predominantly to the Korsmeyer-Peppas kinetics model.Discussion: This study provides a foundational framework for modulating kartogenin release dynamics, a critical consideration for optimizing therapeutic efficacy and minimizing adverse effects in cartilage tissue engineering applications.

AI identifies potent inducers of breast cancer stem cell differentiation based on adversarial learning from gene expression data.

Cancer stem cells (CSCs) are a subpopulation of cancer cells within tumors that exhibit stem-like properties and represent a potentially effective therapeutic target toward long-term remission by means of differentiation induction. By leveraging an artificial intelligence approach solely based on transcriptomics data, this study scored a large library of small molecules based on their predicted ability to induce differentiation in stem-like cells. In particular, a deep neural network model was trained using publicly available single-cell RNA-Seq data obtained from untreated human-induced pluripotent stem cells at various differentiation stages and subsequently utilized to screen drug-induced gene expression profiles from the Library of Integrated Network-based Cellular Signatures (LINCS) database. The challenge of adapting such different data domains was tackled by devising an adversarial learning approach that was able to effectively identify and remove domain-specific bias during the training phase. Experimental validation in MDA-MB-231 and MCF7 cells demonstrated the efficacy of five out of six tested molecules among those scored highest by the model. In particular, the efficacy of triptolide, OTS-167, quinacrine, granisetron and A-443654 offer a potential avenue for targeted therapies against breast CSCs.

Abstract A161: A differentiation based therapeutic approach for KMT2A rearranged leukemia in infants and children

Abstract Introduction: Translocations in the KMT2A gene (KMT2A-r) in leukemia is an aggressive oncodriver. Currently, few therapeutic options are available for these patients and novel therapeutic strategies are urgently needed to improve outcomes. Characteristically, leukemic cells undergo a differentiation block at an early stage of development and agents that restore terminal differentiation represent an effective therapeutic strategy. Based on the initial reports that the inhibition of the Menin-KMT2A fusion is a potent inducer of leukemic cell differentiation, we sought to study a novel treatment approach to enhance the current treatment outcomes in KMT2A-r leukemia. Methods: Pediatric leukemia cells with and without KMT2A-r were studied. Differentiation was induced by the Menin inhibitor MI-463 for 12 days, followed by assessment of differentiation on day 6 and day 12 by Giemsa-Wright staining and visualization using the Infinity analyzer software. In the next set of experiments, the differentiated and undifferentiated cells were treated with common anti-leukemic chemotherapeutic agents and the induction of apoptosis and target modulation analyses were carried out by Annexin/PI staining and immunoblotting. The differentiated cells were also treated with a panel of 220 FDA approved agents, targeting diverse oncogenic pathways to generate a sensitivity signature specific to the differentiated cells. Gene expression array studies were used to identify differentially expressed genes in the two cell populations. Results: The KMT2A-r leukemia cells showed increased dose dependent sensitivity to MI-463. The highest drug sensitivity was exhibited by the infant leukemia cell line MV4-11. At 1µM, no significant loss of cell viability was noted but the rate of proliferation was significantly decreased compared to DMSO control, providing optimal conditions for differentiation. Leukemic cells cultured with MI-463 showed characteristic morphological changes of differentiation including vacuolated cytoplasm, kidney shaped nuclei and decreased nuclei to cytoplasmic ratio. Importantly, we found that the differentiated cells showed a similar chemosensitivity pattern but the sensitivity to AraC was greatly reduced. For example, in MV4-11 cells, IC50 was 170 nM compared to 8 nM in undifferentiated cells. Directed drug screen and differential gene expression studies identified specific growth and survival pathways that can be positively targeted in differentiated cells. Discussion: Data presented demonstrate the activity of MI-463 to induce cell death as well as to initiate differentiation in KMT2A-r cells. We further characterize the sensitivity profile of current chemotherapeutic agents for multi-agent treatment regimens and identify novel agents to optimize future treatment protocols. Differential gene expression analyses were used to understand the potential mechanisms of therapeutic vulnerabilities in these cells. Our results provide the first preclinical evidence to select agents and combinations to improve the outcomes in KMT2A-r leukemia in children. Citation Format: Ritul Sharma, Aru Narendran. A differentiation based therapeutic approach for KMT2A rearranged leukemia in infants and children [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A161.

Early induction of Hes1 by bone morphogenetic protein 9 plays a regulatory role in osteoblastic differentiation of a mesenchymal stem cell line.

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.

The plasticity of fibroblasts: A forgotten player in the aging process.

Tissue-resident fibroblasts are mesenchymal cells which possess an impressive plasticity in their ability to modify their properties according to the requirements of the microenvironment. There are diverse subgroups of fibroblast phenotypes associated with different tissue pathological conditions, e.g., cancers, wound healing, and many fibrotic and inflammatory conditions. The heterogeneous phenotypes can be subdivided into fibrogenic and non-fibrogenic, inflammatory and immunosuppressive subtypes as well as cellular senescent subsets. A major hallmark of activated fibroblasts is that they contain different amounts of stress fibers combined with α-smooth muscle actin (α-SMA) protein, i.e., commonly this phenotype has been called the myofibroblast. Interestingly, several stresses associated with the aging process are potent inducers of myofibroblast differentiation, e.g., oxidative and endoplasmic reticulum stresses, extracellular matrix (ECM) disorders, inflammatory mediators, and telomere shortening. Accordingly, anti-aging treatments with metformin and rapamycin inhibited the differentiation of myofibroblasts in tissues. There is evidence that the senescent phenotype induced in cultured fibroblasts does not represent the phenotype of fibroblasts in aged tissues. Considering the versatile plasticity of fibroblasts as well as their frequency and structural importance in tissues, it does seem that fibroblasts are overlooked players in the aging process.

Intratumoral IL15 Improves Efficacy of Near-Infrared Photoimmunotherapy.

IL15 is a potent inducer of differentiation and proliferation of CD8+ T and natural killer (NK) cells, making it a promising candidate for cancer immunotherapy. However, limited efficacy of systemic monotherapy utilizing intravenous IL15 suggests the needs for alternative routes of administration or combination treatment with other therapies. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective anticancer treatment that elicits a massive release of tumor antigens and immunogenic signals. Here, we investigated whether intratumoral IL15 can enhance the effectiveness of cancer cell-targeted NIR-PIT using syngeneic murine tumor models. Intratumoral injection of IL15 was more effective than intraperitoneal IL15 in vivo in suppressing tumor growth and inducing intratumoral immune responses. When the efficacy of CD44-targeted NIR-PIT was compared in vivo between IL15-secreting MC38 (hIL15-MC38) and parental MC38 tumors, the hIL15-MC38/NIR-PIT group showed the best tumor growth inhibition and survival. In addition, the hIL15-MC38/NIR-PIT group showed significant dendritic cell maturation and significant increases in the number and Granzyme B expression of tumor-infiltrating CD8+ T, NK, and natural killer T cells compared with the treated parental line. Furthermore, intratumoral IL15 injection combined with CD44-targeted NIR-PIT showed significant tumor control in MC38 and Pan02-luc tumor models. In bilateral tumor models, CD44-targeted NIR-PIT in hIL15-MC38 tumors significantly suppressed the growth of untreated MC38 tumors, suggesting abscopal effects. Mice that achieved complete response after the combination therapy completely rejected later tumor rechallenge. In conclusion, local IL15 administration synergistically improves the efficacy of cancer cell-targeted NIR-PIT probably by inducing stronger anticancer immunity, indicating its potential as an anticancer treatment strategy.

Association of Circulating Fibrocytes With Fibrostenotic Small Bowel Crohn's Disease.

Fibrocytes are hematopoietic cells with features of mesenchymal cells found in the circulation and inflammatory sites implicated in promoting fibrosis in many fibroinflammatory diseases. However, their role(s) in the development of intestinal fibrosis is poorly understood. Here, we investigated a potential role of fibrocytes in the development of fibrosis in Crohn's disease (CD) and sought factors that may impact their development and function. Plasma and mononuclear cells were collected from patients with and without fibrostenotic CD. Fibrocytes defined as CD11b+, CD34+, and Collagen 1+ were correlated with clinical assessments of fibrosis, including evaluation using intestinal ultrasound. We measured the levels of relevant circulating molecules via Luminex and studied the effect of patient plasma proteins on fibrocyte differentiation. Fibrocyte numbers were increased in CD patients with stricturing Crohn's disease compared with patients with an inflammatory phenotype (P = .0013), with strong correlation between fibrocyte numbers and acoustic radiation force impulse (ARFI), a measure of bowel elasticity on intestinal ultrasound (R = .8383, P = .0127). Fibrostenotic plasma was a more potent inducer of fibrocyte differentiation in both primary human monocytes and cell line and contained increased levels of cytokines implicated in fibrocyte differentiation compared with plasma from inflammatory patients. Interestingly, increased fibrocyte numbers at time of ultrasound were associated with escalation of medical therapy and endoscopic/surgical management of small bowel strictures at 30 months follow-up. Circulating fibrocytes strongly correlate with fibrostenotic disease in CD, and they may serve as predictors for escalation of medical +/- surgical therapy.

Hexane Extract of Chloranthus japonicus Increases Adipocyte Differentiation by Acting on Wnt/β-Catenin Signaling Pathway.

Chloranthus japonicus has been heavily investigated for the treatment of various diseases. This paper attempts to show that Chloranthus japonicus can modulate adipocyte differentiation of preadipocytes. To establish this, we investigated the effects of Chloranthus japonicus extract in peroxisome proliferator-activated receptor γ (PPARγ) expression, adipogenesis, and the underlying molecular mechanisms in C3H10T1/2 and 3T3-L1 cells. Our data showed that Chloranthus japonicus methanol extract increased lipid accumulation and promoted adipocyte differentiation. Further studies on the fractionation with various solvents led to the identification of Chloranthus japonicus hexane extract (CJHE) as the most potent inducer of adipocyte differentiation. CJHE consistently increased lipid accumulation and adipocyte marker expression including Pparγ and it acted during the early stages of adipocyte differentiation. Mechanistic studies revealed that CJHE and a Wnt inhibitor similarly stimulated adipogenesis and were active in Wnt-selective reporter assays. The effects of CJHE were inhibited by Wnt3a protein treatment and were significantly blunted in β-catenin-silenced cells, further suggesting that CJHE acted on Wnt pathways to promote adipogenesis. We also showed that Chloranthus japonicus extracts generated from different plant parts similarly promoted adipocyte differentiation. These results identified Chloranthus japonicus as a pro-adipogenic natural product and suggest its potential use in metabolic syndrome.

Whole transcriptome sequencing and integrated network analysis elucidates the effects of 3,8-Di-O-methylellagic acid 2-O-glucoside derived from Sanguisorba offcinalis L., a novel differentiation inducer on erythroleukemia cells

Acute erythroid leukemia (AEL) is a rare and aggressive hematologic malignancy with no specific treatment. Sanguisorba officinalis L. (S. officinalis), a well-known traditional Chinese medicine, possesses potent anticancer activity. However, the active components of S. officinalis against AEL and the associated molecular mechanisms remain unknown. In this study, we predicted the anti-AML effect of S. officinalis based on network pharmacology. Through the identification of active components of S. officinalis, we found that 3,8-Di-O-methylellagic acid 2-O-glucoside (DMAG) not only significantly inhibited the proliferation of erythroleukemic cell line HEL, but also induced their differentiation to megakaryocytes. Furthermore, we demonstrated that DMAG could prolong the survival of AEL mice model. Whole-transcriptome sequencing was performed to elucidate the underlying molecular mechanisms associated with anti-AEL effect of DMAG. The results showed that the total of 68 miRNAs, 595 lncRNAs, 4030 mRNAs and 35 circRNAs were significantly differentially expressed during DMAG induced proliferation inhibition and differentiation of HEL cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the differentially expressed miRNAs, lncRNAs, mRNAs and circRNAs were mainly involved in metabolic, HIF-1, MAPK, Notch pathway and apoptosis. The co-expression networks showed that miR-23a-5p, miR-92a-1–5p, miR-146b and miR-760 regulatory networks were crucial for megakaryocyte differentiation induced by DMAG. In conclusion, our results suggest that DMAG, derived from S. officinalis might be a potent differentiation inducer of AEL cells and provide important information on the underlying mechanisms associated with its anti-AEL activity.

Effect of vorinostat on INK4 family and HDACs 1, 2, and 3 in pancreatic cancer and hepatocellular carcinoma.

Background and purpose:In mammalian cells, several distinct surveillance systems, named cell cycle checkpoints, can interrupt normal cell-cycle progression. The cyclin-dependent kinases are negatively regulated by proteins of cyclin-dependent kinases inhibitors comprising INK4 and Cip/Kip families. Histone deacetylation induced by histone deacetylases (HDACs) inactivates the INK4 and Cip/Kip families lead to cancer induction. HDAC inhibitors (HDACIs) have been indicated to be potent inducers of differentiation, growth arrest, and apoptotic induction. Vorinostat (suberoylanilide hydroxamic acid, SAHA), as an HDACI, is reported to be useful in various cancers. Previously, we reported the effect of trichostatin A on hepatocellular carcinoma and also vorinostat on colon cancer cell lines. The current study was aimed to investigate the effect of vorinostat on p16INK4a, p14ARF, p15INK4b, and class I HDACs 1, 2, and 3 gene expression, cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and hepatocellular carcinoma LCL-PI 11 cell lines.Experimental approach:The AsPC-1 and LCL-PI 11 cell lines were cultured and treated with vorinostat. To determine, viability, apoptosis, and the relative expression level of p16INK4a, p14ARF, p15INK4b, class I HDACs 1, 2, and 3 genes, MTT assay, cell apoptosis assay, and RT-qPCR were performed, respectively.Findings/Results:Vorinostat significantly inhibited cell growth, induced apoptosis, increased p16INK4a, p14ARF, p15INK4b, and decreased class I HDACs 1, 2, and 3 gene expression.Conclusion and implications:Vorinostat can reactivate the INK4 family through inhibition of class I HDACs 1, 2, and 3 genes activity.

PRMT1 inhibition induces differentiation of colon cancer cells

Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.

Effect of Nicotinamide, 1-Methylnicotinamide, and N’-Methylnicotinamide on Erythroid Colony Formation and γ-Globin Expression in Cultured Baboon CD34+ Cells

Pharmacological treatments designed to increase Fetal Hemoglobin (HbF) levels offer great promise to alleviate the symptoms and improve the lifespan of the vast numbers of patients afflicted with sickle cell disease (SCD) and β-thalassemia. Hydroxyurea can increase HbF, but a large fraction of patients with SCD do not respond to the drug. DNMT1 and LSD1 inhibitors are the most powerful drugs to increase HbF but are limited by side effects that include neutropenia, thrombophilia and/or thrombocytopenia. The development of new, more effective, and safer pharmacological strategies to augment HbF levels in the blood thus continues to be an important goal. Previous studies have shown that γ-globin gene expression is dynamically regulated during erythroid differentiation (Papayannopoulou et al PNAS 74:2923-2927, 1977). The proportion of γ-globin gene expression is higher at earlier stages (BFUe) of erythroid differentiation than at more advanced stages (CFUe). Therefore, we suggest the hypothesis that expansion of primitive, less differentiated progenitors might favor increased γ-globin, particularly when combined other HbF-inducing drugs. To investigate this hypothesis, we have tested whether nicotinamide (NAM), the major NAM metabolite 1-methylnicotinamide (1-mNAM), and N'-methylnicotinamide (N'-mNAM), a chemical derivative of NAM, can foster expansion of erythroid colony-forming cells (BFUe and CFUe) and increase γ-globin expression of cultured baboon CD34+ cells. Previous observations have shown that NAM facilitates in vitro expansion of cord blood CD34+ cells and enhanced long term engraftment in transplanted recipients (Horwitz et al J Clin Invest 124:3121, 2014). Contrasting effects of NAM, 1-mNAM, and N'-mNAM on differentiation and proliferation of the murine erythroleukemia cell line (MEL) have been previously reported (Terada et al PNAS 76:6414, 1979; Kuykendall et al Toxicol In Vitro 21:1656, 2007). While both NAM and N'-mNAM induced MEL cell differentiation, N'-mNAM was far more potent. In contrast, 1-mNAM increased cell proliferation, reduced spontaneous differentiation, and blocked differentiation induced by NAM and N'-mNAM. The effect of all three forms of NAM was examined using bone marrow (BM) CD34+ cells from a pre-clinical non-human primate large animal model. To test the effect of NAM, 1-mNAM, and N'-mNAM on expansion of erythroid colony-forming cells, the agents (5mM) were added to liquid cultures of baboon CD34+ bone marrow cells previously expanded for 5 days in serum-free expansion media (SFEM). Colony assays were performed on d8. In two experiments total erythroid colonies (BFUe and CFUe) were 2 fold higher in cultures treated with 1-mNAM compared to untreated controls (p<0.05) while no effect was observed in cultures treated with NAM. No colonies were observed in cultures treated with N'-mNAM (Figure 1A). Observation of Wright's stained cytospin preparations showed extensive erythroid differentiation on d8 in cells treated with N'-mNAM (Figure 1B). The effect of NAM, 1-mNAM, and N'-NAM on γ-globin expression was tested in baboon CD34+ cells grown in co-culture with the AFT024 cell line. NAM, 1-mNAM, or N'-mNAM (5mM) were added to cultures on d7. Expression of γ- and β-globin mRNA was measured by RT-PCR on d17. Increased γ-globin expression (0.57±0.04 γ/γ+β)) was observed in cells treated with N'-mNAM on d7 compared to untreated controls (0.20±0.09 γ/γ+β; p<0.001). NAM and 1-mNAM had no significant effect on γ-globin gene expression (Figure 1C). These results thus show that while erythroid colonies are increased by 1-mNAM, N'-mNAM is a potent inducer of erythroid differentiation and increases γ-globin expression in primary cultures of baboon CD34+ cells. In conclusion, 1-mNAM and N'-mNAM have contrasting effects on erythroid differentiation in primary baboon CD34+ cell cultures, confirming previous experiments in the MEL cell line. Future experiments are planned to test the effect of these agents on HbF in the baboon. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Design, synthesis, and biological evaluation of spiroindolines as novel inducers of oligodendrocyte progenitor cell differentiation—Use of a conformation-based hypothesis to facilitate compound design

Inducing oligodendrocyte progenitor cell (OPC) differentiation is a novel therapeutic strategy for the treatment of demyelinating diseases such as multiple sclerosis (MS). In the preceding article, we detailed the discovery of compound 1, a potent inducer of OPC differentiation possessing a characteristic spiroindoline structure. Also, we found that N-methylation and des-carbonyl compound 1 (4) led to a loss in potency. Herein, we describe our investigations of a conformation-based hypothesis for OPC differentiation activity based on the preferred conformation of the spiro core, and further structure–activity relationship (SAR) exploration led to the identification of 6-CF3 derivative 8, which was more potent compared to compound 1.

Belinostat resolves skin barrier defects in atopic dermatitis by targeting the dysregulated miR-335:SOX6 axis

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Skin barrier defects contribute to disease initiation and development; however, underlying mechanisms remain elusive. To understand the underlying cause of barrier defect, we investigated aberrant expression of specific microRNAs (miRNAs) in AD. Delineating the molecular mechanism of dysregulated miRNA network, we focused on identification of specific drugs that can modulate miRNA expression and repair the defective barrier in AD. A screen for differentially expressed miRNAs between healthy skin and AD lesional skin resulted in the identification of miR-335 as the most consistently downregulated miRNA in AD. Using in silico prediction combined with experimental validation, we characterized downstream miR-335 targets and elucidated the molecular pathways by which this microRNA maintains epidermal homeostasis in healthy skin. miR-335 was identified as a potent inducer of keratinocyte differentiation; it exerts this effect by directly repressing SOX6. By recruiting SMARCA complex components, SOX6 suppresses epidermal differentiation and epigenetically silences critical genes involved in keratinocyte differentiation. In AD lesional skin, miR-335 expression is aberrantly lost. SOX6 is abnormally expressed throughout the epidermis, where it impairs skin barrier development. We demonstrate that miR-335 is epigenetically regulated by histone deacetylases; a screen for suitable histone deacetylase inhibitors identified belinostat as a candidate drug that can restore epidermal miR-335 expression and rescue the defective skin barrier in AD. Belinostat is of clinical significance not only as a candidate drug for AD treatment, but also as a potential means of stopping the atopic march and further progression of this systemic allergic disease.

Platelet-poor plasma of athletes is a potent inducer of myogenic differentiation of C2C12 myoblasts

Introduction. Blood products, i.e. platelet rich plasma (PRP), leukocyte-poor plasma (PRP) and platelet poor plasma (PPP), have previously been used to improve muscle regeneration. In this study, six months? frozen-stored PPP of individuals who practiced different types of physical exercise was analysed; it could steer mouse C2C12 myoblast cells towards proliferation, migration and myogenic differentiation, and it could affect the morphology/shape of myotubes. Materials and Methods. PPP of male Olympic weightlifters, football players and professional folk dancers, aged 15-19, was collected 12 h post-training and stored for 6 months at -20?C. C2C12 cell proliferation was assessed by MTT test, motility by scratch assay, myogenic differentiation by myotube formation and gelatinase activity by gel-zymography. Results and Conclusions. PPP induced proliferation and migration of C2C12 cells. Proliferative capacity was as follows: weightlifters > dancers > football players; mean migratory capacity was: weightlifters = dancers > football players. PPP induced formation of myotubes; significant inter-individual variations were detected: PPP from weightlifters induced formation of round myotubes, and PPP from football players and dancers induced formation of elongated myotubes. The mean myotube area was as follows: football players > dancers > weightlifters. PPP gelatinolytic activity was observed; it was negatively correlated with C2C12 myoblast proliferation. These results provide general but distinct evidence that PPP of individuals practicing certain types of exercise can specifically modify myoblast morphology/function. This is significant for explaining physiological responses and adaptations to exercise. In conclusion, long-term, frozen-stored PPP preserves its potential to modify myoblast morphology and function.

MiR-17 and miR-19 cooperatively promote skeletal muscle cell differentiation

Skeletal myogenesis is a highly coordinated process that involves cell proliferation, differentiation and fusion controlled by a complex gene regulatory network. The microRNA gene cluster miR-17–92 has been shown to be related to this process; however, the exact role of each cluster member remains unclear. Here, we show that miR-17 and miR-20a could effectively promote the differentiation of both C2C12 myoblasts and primary bovine satellite cells. In contrast, miR-18a might play a negative role in C2C12 cell differentiation, while miR-19 and miR-92a had little influence. Transcriptome and target analyses revealed that miR-17 could act on Ccnd2, Jak1 and Rhoc genes that are critical for cell proliferation and/or fusion. Notably, the addition of miR-19 could reverse the lethal effect of miR-17 and could thus facilitate the maturation of myotubes. Furthermore, by co-injecting the lentiviral shRNAs of miR-17 and miR-19 into mouse tibialis anterior muscles, we demonstrated the wound healing abilities of the two miRNAs. Our findings indicate that in combination with miR-19, miR-17 is a potent inducer of skeletal muscle differentiation.

Thiourea derivatives induce fetal hemoglobin production in-vitro: A new class of potential therapeutic agents for β-thalassemia

Fetal hemoglobin (HbF) induction is a cost-effective therapeutic approach for the treatment of β-hemoglobinopathies like β-thalassemia and sickle cell anemia. The present study discusses the potential of thiourea derivatives as new class of compounds that induce the fetal hemoglobin production. HbF inducing effect of thiourea derivatives was studied using experimental cell system, the human erythroleukemic K562 cell line. Erythroid induction of K562 cells was studied by the benzidine/H2O2 reaction, total hemoglobin production was estimated by plasma hemoglobin assay kit, and γ-globin gene expression by RT-qPCR, whereas fetal hemoglobin production was estimated by flow cytometry and immunofluorescence microscopy. The results indicated that newly synthesized thiourea derivative are potent inducers of erythroid differentiation of K562 cells with an increased γ-globin gene expression and fetal hemoglobin production. Moreover, these compounds showed no cytotoxic effect and inhibition on K562 cells at HbF inducing concentrations. It is important to note that hydroxyurea is a cytotoxic chemotherapeutic agent and have deleterious side effects, reflecting the need to identify new safe and effective HbF induces. These results signify thiourea derivatives as promising HbF inducers, with the potential to be studied against hematological disorders, including β-thalassemia and sickle cell anemia.

Insulin-like growth factor-I promotes hepatic differentiation of human adipose tissue-derived stem cells.

There are numerous studies which provide support for the use of human adipose tissue-derived stem cells (hASCs) to generate hepatocyte-like cells. However, the produced cells exhibit only a certain level of differentiation, mainly due to inefficient induction conditions. Therefore, based on the important role of insulin-like growth factor (IGF-I) in hepatic function and development, in the current study we evaluated the differentiation efficacy of the mentioned factor to induce hASCs into functional hepatocyte-like cells. To investigate this, using a two-step protocol, hASCs were treated with a combination of HGF, Dex, and OSM in the presence or absence of IGF-I up to 21 days. Hepatic differentiation was evaluated by analyzing specific hepatocyte markers at different time points of differentiation induction. Increased expression of hepatocyte-specific genes including ALB, AFP, CK18, and HNF4a, downregulation of bile duct cells marker (CK19), the higher number of ALB positive cells, increased urea production together with higher glycogen deposit was observed upon the treatment of hASCs with the induction medium containing IGF-I compared to the other treatment. In conclusion, our findings suggest IGF-I as a potent inducer of hepatic differentiation of hASCs and its potential to generate more functional hepatocyte-like cells.

BMP9-induced osteoblastic differentiation requires functional Notch signaling in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into multiple lineages including osteoblastic lineage. Osteogenic differentiation of MSCs is a cascade that recapitulates most, if not all, of the molecular events occurring during embryonic skeletal development, which is regulated by numerous signaling pathways including bone morphogenetic proteins (BMPs). Through a comprehensive analysis of the osteogenic activity, we previously demonstrated that BMP9 is the most potent BMP for inducing bone formation from MSCs both in vitro and in vivo. However, as one of the least studied BMPs, the essential mediators of BMP9-induced osteogenic signaling remain elusive. Here we show that BMP9-induced osteogenic signaling in MSCs requires intact Notch signaling. While the expression of Notch receptors and ligands are readily detectable in MSCs, Notch inhibitor and dominant-negative Notch1 effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic bone formation in vivo. Genetic disruption of Notch pathway severely impairs BMP9-induced osteogenic differentiation and ectopic bone formation from MSCs. Furthermore, while BMP9-induced expression of early-responsive genes is not affected by defective Notch signaling, BMP9 upregulates the expression of Notch receptors and ligands at the intermediate stage of osteogenic differentiation. Taken together, these results demonstrate that Notch signaling may play an essential role in coordinating BMP9-induced osteogenic differentiation of MSCs.Bone morphogenetic protein (BMP) 9 is a potent inducer of osteogenic differentiation from mesenchymal stem cells, but the mediators of BMP9-induced osteogenesis remain elusive. Here, the authors show that inhibition of Notch1 signaling effectively diminishes BMP9-induced osteogenesis. Genetic disruption of Notch pathway severely impairs BMP9-induced bone formation. Thus, these results demonstrate that Notch signaling may play an essential role in coordinating osteogenic differentiation.

Autophagy is Required to Regulate Mitochondria Renewal, Cell Attachment, and All-trans–Retinoic Acid–Induced Differentiation in NB4 Acute Promyelocytic Leukemia Cells

All-trans-retinoic acid (ATRA) is a potent inducer of cellular differentiation, growth arrest, and apoptosis as well as a front-line therapy for acute promyelocytic leukemia (APL). The present study provides evidence that induction of autophagy is required for ATRA to induce differentiation of APL (NB4) cells into granulocytes. ATRA treatment causes ~12-fold increase in the number of acidic vesicular organelles and induces marked up-regulation of LC3-II, autophagy-related 5 (ATG5), and Beclin-1. Transmission electron microscopy (TEM) revealed a decrease in mitochondria and ATRA-induced differentiation. To determine the role of autophagy in the differentiation of APL, we knocked down ATG5 in NB4 cells to find that ATRA-induced differentiation is significantly inhibited during ATG5 knock down in cells, indicating the role of autophagy in differentiation of APL. Further experiments revealed restriction of autophagy during ATRA-induced differentiation and inhibition of tissue transglutaminase 2 (TG2) and phospho-focal adhesion kinase (p-FAK), which are known to have roles in differentiation and cell attachment. We examinedexpression of Beclin-1 and B-cell lymphoma-2 (Bcl-2) and levels of mechanistic target of rapamycin (mTOR) after ATRA treatment. ATRA inhibits Bcl-2, up-regulates Beclin-1 expression, and reduces induction of mTOR activation/phosphorylation in NB4 cells. Our results reveal that autophagy has roles in regulation of differentiation, mitochondria elimination, and cell attachment during ATRA-induced APL differentiation.