Forced expression of CD200 improves the differentiation capability and immunoregulatory functions of mesenchymal stromal cells.

OBJECTIVE: In order to identify specific mesenchymal stromal cell (MSC) populations with enhanced therapeutic efficacy, we evaluated the functional changes associated with the stable expression of CD200, which is associated with immune regulatory function and osteogenic differentiation, in human bone marrow-derived MSCs (CD200/MSCs). RESULTS: We detected significantly greater osteogenesis and chondrogenesis in CD200/MSCs than in mock-transfected MSCs. In addition, the immune regulatory function of MSCs in mixed lymphocyte reactions was enhanced by CD200 gene transfection. In CD200/MSCs, the secretion of inflammatory cytokines, i.e., IL-6 and IL-8, was reduced, and levels of the anti-inflammatory factors IL-10, FOXP3, and indoleamine 2,3-dioxygenase 1 were elevated. Finally, CD200 transfection increased the stemness of MSCs, as evidenced by greater colony numbers in colony-forming unit fibroblast assays and analyses of NANOG and OCT-4 expression. CONCLUSIONS: These results suggest that CD200/MSCs have therapeutic applications, and further in-depth research should focus on the development of a clinically applicable cell-based therapeutic strategy.

Enhancement of human mesenchymal stem cell differentiation by combination treatment with 5-azacytidine and trichostatin A.

OBJECTIVE: To enhance the differentiation of mesenchymal stem cells (MSCs) and their epigenetic status by modification using hypomethylating agents (HMAs) and histone deacetylase inhibitors (HDACs). RESULTS: Treatment with 5-azacytidine or 5-azacytidine plus trichostatin A (TSA) increased expression of Runx-2, BDNF and Sox-9 compared with the control or TSA groups. Maximal increases of 4.1-, 4.5-, and 8.3-fold in Runx-2, BDNF, and Sox-9 transcript levels, respectively, were observed in the 5-azacytidine plus TSA group. Similar to the expression pattern of key regulatory molecules, differentiation to each lineage was also enhanced considerably in the 5-azacytidine or in the 5-azacytidine plus TSA groups. Quantitative analyses at the protein level showed 8.9-, 26.8-, 27.9-, and 28.5-fold upregulation of osterix, MAP-2, nestin, and type II collagen), respectively. CONCLUSION: HMAs and HDACs enhanced in vitro differentiation of MSCs, which was maximized when the two drugs were combined, with HMA having the dominant effect.

Development of Mesenchymal Stem Cell Encoded with Myogenic Gene for Treating Radiation-Induced Muscle Fibrosis.

Radiation therapy (RT) is a typical treatment for head and neck cancers. However, prolonged irradiation of the esophagus can cause esophageal fibrosis due to increased reactive oxygen species and proinflammatory cytokines. The objective of this study was to determine whether myogenic gene-transfected mesenchymal stem cells (MSCs) could ameliorate damage to esophageal muscles in a mouse model of radiation-induced esophageal fibrosis. We cloned esophageal myogenic genes (MyoD, MyoG, and Myf6) using plasmid DNA. Afterward, myogenic genes were transfected into Human Mesenchymal Stem Cells (hMSCs) using electroporation. Gene transfer efficiency, stemness, and myogenic gene profile were examined using flow cytometry, quantitative polymerase chain reaction, and RNA sequencing. In vivo efficacy of gene-transfected hMSCs was demonstrated through histological and gene expression analyses using a radiation-induced esophageal fibrosis animal model. We have confirmed that the gene transfer efficiency was high (∼75%). Pluripotency levels in gene-transfected MSCs were significantly decreased compared with those in the control (vector). Particularly, myogenesis-related genes such as OAS2, OAS3, and HSPA1A were overexpressed in the group transfected with three genes. At 4 weeks after injection, it was found that thickness collagen layer and esophageal muscle in MSCs transfected with all three genes were significantly reduced compared to those in the saline group. Muscularis mucosa was observed prominently in the gene combination group. Moreover, expression levels of myogenin, Myf6, calponin, and SM22α known to be specific markers of esophageal muscles tended to increase in the group transfected with three genes. Therefore, using gene-transfected MSCs has the potential as a promising therapy against radiation-induced esophageal fibrosis.

c-Src activation through a TrkA and c-Src interaction is essential for cell proliferation and hematological malignancies.

Although the kinase receptor TrkA may play an important role in acute myeloid leukemia (AML), its involvement in other types of leukemia has not been reported. Furthermore, how it contributes to leukemogenesis is unknown. Here, we describe a molecular network that is important for TrkA function in leukemogenesis. We found that TrkA is frequently overexpressed in other types of leukemia such as acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS) including AML. In addition, TrkA was overexpressed in patients with MDS or secondary AML evolving from MDS. TrkA induced significant hematological malignancies by inducing PLK-1 and Twist-1, and enhanced survival and proliferation of leukemia, which was correlated with activation of the phosphatidylinositol 3-kinase/Akt/mTOR pathway. Moreover, endogenous TrkA associated with c-Src complexes was detected in leukemia. Suppression of c-Src activation by TrkA resulted in markedly decreased expression of PLK-1 and Twist-1 via suppressed activation of Akt/mTOR cascades. These data suggest that TrkA plays a key role in leukemogenesis and reveal an unexpected physiological role for TrkA in the pathogenesis of leukemia. These data have important implications for understanding various hematological malignancies.

A genetic link between cold responses and flowering time through FVE in Arabidopsis thaliana.

Cold induces expression of a number of genes that encode proteins that enhance tolerance to freezing temperatures in plants. A cis-acting element responsive to cold and drought, the C-repeat/dehydration-responsive element (C/DRE), was identified in the Arabidopsis thaliana stress-inducible genes RD29A and COR15a and found in other cold-inducible genes in various plants. C/DRE-binding factor/DRE-binding protein (CBF/DREB) is an essential component of the cold-acclimation response, but the signaling pathways and networks are mostly unknown. Here we used targeted genetic approach to isolate A. thaliana mutants with altered cold-responsive gene expression (acg) and identify ACG1 as a negative regulator of the CBF/DREB pathway. acg1 flowered late and had elevated expression of FLOWERING LOCUS C (FLC), a repressor of flowering encoding a MADS-box protein. We showed that acg1 is a null allele of the autonomous pathway gene FVE. FVE encodes a homolog of the mammalian retinoblastoma-associated protein, a component of a histone deacetylase (HDAC) complex involved in transcriptional repression. We also showed that plants sense intermittent cold stress through FVE and delay flowering with increasing expression of FLC. Dual roles of FVE in regulating the flowering time and the cold response may have an evolutionary advantage for plants by increasing their survival rates.

Therapeutic Effects and Underlying Mechanism of SOCS-com Gene-Transfected ADMSCs in Pressure Ulcer Mouse Models.

Although the proportion of ulcer patients with medical problems among the elderly has increased with the extension of human life expectancy, treatment efficiency is drastically low, incurring substantial social costs. MSCs have independent regeneration potential, making them useful in clinical trials of difficult-to-treat diseases. In particular, ADMSCs are promising in the stem cell therapy industry as they can be obtained in vast amounts using non-invasive methods. Furthermore, studies are underway to enhance the regeneration potential of ADMSCs using cytokines, growth factors, and gene delivery to generate highly functional ADMSCs. In this study, key regulators of wound healing, SOCS-1, -3, and -5, were combined to maximize the regenerative potential of ADMSCs in pressure ulcer treatments. After transfecting SOCS-1, -3, -5, and SOCS-com into ADMSCs using a non-viral method, the expression of the inflammatory factors TNF-alpha, INF-gamma, and IL-10 was confirmed. ADMSCs transfected with SOCS-com showed decreased overall expression of inflammatory factors and increased expression of anti-inflammatory factors. Based on these results, we implanted ADMSCs transfected with SOCS-com into a pressure ulcer mouse model to observe their subsequent wound-healing effects. Notably, SOCS-com improved wound closure in ulcers, and reconstruction of the epidermis and dermis was observed. The healing mechanism of ADMSCs transfected with SOCS-com was examined by RNA sequencing. Gene analysis results confirmed that expression changes occurred in genes of key regulators of wound healing, such as chemokines, MMP-1, 9, CSF-2, and IL-33, and that such genetic changes enhanced wound healing in ulcers. Based on these results, we demonstrate the potential of ADMSCs transfected with SOCS-com as an ulcer treatment tool.

Undifferentiated hematopoietic cells are characterized by a genome-wide undermethylation dip around the transcription start site and a hierarchical epigenetic plasticity.

Evidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from a genome-wide comparison of CpG methylation in human CD34(+) and CD34(-) cells, we identified a characteristic undermethylation dip around the transcription start site of promoters and an overmethylation of flanking regions in undifferentiated CD34(+) cells. This "bivalent-like" CpG methylation pattern around the transcription start site was more prominent in genes not associated with CpG islands (CGI(-)) than CGI(+) genes. Undifferentiated hematopoietic cells also exhibited dynamic chromatin associated with active transcription and a higher turnover of histone acetylation than terminally differentiated cells. Interestingly, inhibition of chromatin condensation by chemical treatment (5-azacytidine, trichostatin A) enhanced the self-renewal of "stimulated" HSCs in reconstituting bone marrows but not "steady-state" HSCs in stationary phase bone marrows. In contrast, similar treatments on more mature cells caused partial phenotypic dedifferentiation and apoptosis at levels correlated with their hematopoietic differentiation. Taken together, our study reveals that the undifferentiated state of hematopoietic cells is characterized by a unique epigenetic signature, which includes dynamic chromatin structures and an epigenetic plasticity that correlates to level of undifferentiation.

Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture.

The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P<0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P<0.05) and that of type I collagen significantly decreased (P<0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.

Clinical implications of copy number alteration detection using panel-based next-generation sequencing data in myelodysplastic syndrome.

Recent advancements in next-generation sequencing (NGS) technologies allow the simultaneous identification of targeted copy number alterations (CNAs) as well as somatic mutations using the same panel-based NGS data. We investigated whether CNAs detected by the targeted NGS data provided additional clinical implications, over somatic mutations, in myelodysplastic syndrome (MDS). Targeted deep sequencing of 28 well-known MDS-related genes was performed for 266 patients with MDS. Overall, 215 (80.8 %) patients were found to have at least one somatic mutation; 67 (25.2 %) had at least one CNA; 227 (85.3 %) had either a somatic mutation or CNA; and 12 had CNA without somatic mutations. Considering the clinical variables and somatic mutations alone, multivariate analysis demonstrated that sex, revised International Prognostic Scoring System (IPSS-R), and NRAS and TP53 mutations were independent prognostic factors for overall survival. For AML-free survival, these factors were sex, IPSS-R, and mutations in NRAS, DNMT3A, and complex karyotype/TP53 mutations. When we consider clinical variables along with somatic mutations and CNAs, genetic alterations in TET2, LAMB4, U2AF1, and CBL showed additional significant impact on the survivals. In conclusion, our study suggests that the concurrent detection of somatic mutations and targeted CNAs may provide clinically useful information for the prognosis of MDS patients.

Gene expression signatures associated with sensitivity to azacitidine in myelodysplastic syndromes.

Allogeneic stem cell transplantation is currently the only curative treatment option for myelodysplastic syndromes (MDS). Pre-transplant debulking treatment have been employed for advanced MDS and we previously reported that marrow response (blast ≤ 5%) following the bridging therapy with hypomethylating agent was an independent favorable factor for survival; however, it is still not clear which patients will respond to hypomethylating agent and which genomic features can predict the response. In this study, we performed RNAseq for 23 MDS patients among which 14 (61%) and 9 (39%) patients showed marrow complete remission and primary resistance to azacitidine, respectively. Differential expression-based analyses of treatment-naive, baseline gene expression profiles revealed that molecular functions representing mitochondria and apoptosis were up-regulated in responders. In contrast, we identified genes involved in the Wnt pathway were relatively up-regulated in non-responders. In independent validation cohorts of MDS patients, the expression of gene sets specific to non-responders and responders distinguished the patients with favorable prognosis and those responded to azacitidine highlighting the prognostic and predictive implication. In addition, a systems biology approach identified genes involved in ubiquitination, such as UBC and PFDN2, which may be key players in the regulation of differential gene expression in treatment responders and non-responders. Taken together, identifying the gene expression signature may advance our understanding of the molecular mechanisms of azacitidine and may also serve to predict patient responses to drug treatment.

Effects of decitabine on allogeneic immune reactions of donor lymphocyte infusion via activation of dendritic cells.

BACKGROUND: Successful prevention of post-transplantation relapse after donor lymphocyte infusion (DLI) depends on its capability to mediate an effective graft-versus-leukemia (GVL) response while minimizing DLI-related toxicity, including graft-versus-host disease (GVHD). METHODS: We assessed the effects of decitabine (DEC), a hypomethylating agent, upon allogeneic immune reaction in a murine model of DLI. RESULTS: Significantly greater tumor growth retardation and survival prolongation occurred in mice administered with 1.0 mg/kg DEC for 5 days (DEC-1.0) than in control or DEC-0.1 mice. Upon prompt DEC and DLI co-administration, dendritic cells (DCs) were activated; DEC-1.0/DLI induced severe GVHD, and survival was significantly lower than with DLI alone or DEC-0.1/DLI treatments. IFN-γ and CD28 levels were higher in splenic DCs of DEC-1.0 mice than in those of control mice. Assessment of delayed DLI co-administration with DEC, when IFN-γ levels were normalized to control levels, revealed that DEC-1.0/DLI successfully facilitated tumor management without causing severe GVHD. CONCLUSIONS: Our results suggest that DEC primes allogeneic immune reactions of DLI via DC activation, and GVHD and GVL effects are separable through optimal DLI timing based on DEC-induced increase in IFN-γ expression levels.

Is continuous treatment with transforming growth factor-beta necessary to induce chondrogenic differentiation in mesenchymal stem cells?

This study was conducted to determine whether short-term administration of transforming growth factor (TGF)-beta would be as effective for inducing chondrogenesis in human mesenchymal stem cells (hMSCs) as continuous treatment. Four groups of hMSCs were cultured in a monolayer for 3 days followed by a pellet culture for 3 weeks under various conditions: group A, the control group, no growth factors treated; group B, 5 ng/ml of TGF-beta(2) was treated for 3 days in monolayer culture; group C, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 3 days of pellet culture; group D, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 10 days of pellet culture; group E, 5 ng/ml of TGF-beta(2) was continuously treated throughout the culture period. Glycosaminoglycan contents significantly increased in group E only. Real-time PCR indicated that expression of Sox-9, type II collagen, type II procollagen B and type X collagen increased with longer duration of TGF-beta(2) treatment. The histological findings showed that longer duration of TGF-beta(2) treatment led to significantly better quality of chondrogenesis. This study demonstrated that longer duration of TGF-beta treatment is necessary for effective chondrogenesis in hMSCs from bone marrow.

Enhanced polo-like kinase 1 expression in myelodysplastic syndromes.

BACKGROUND: Cancer is characterized by uncontrolled cellular proliferation, and Polo-like kinase 1 (PLK1), a key regulator of the cell cycle, is overexpressed in many cancers, including acute leukemia and lymphoma. However, the dynamics of PLK1 transcription in myelodysplastic syndromes (MDS) are unknown. This study aimed to investigate the transcript dynamics of PLK1 and determine its role in the pathophysiology of MDS. METHODS: PLK1 mRNA obtained from the bone marrow samples of 67 patients with MDS, 16 patients with secondary acute myeloid leukemia (sAML), and 10 healthy controls were analyzed using quantitative real-time PCR and compared according to various clinical parameters. RESULTS: The median PLK1 expression levels differed slightly, but not significantly, between MDS and sAML patients [661.21 (range, 29.38-8,987.31) vs. 1,462.05 (32.22-5,734.09), respectively], but were significantly higher (P<0.001) than the levels in the healthy controls [19.0 (1.60-49.90)]. Further analyses of PLK1 levels according to the WHO classification of MDS, prognostic risk groups, karyotype risk groups, marrow blast percentage, and depth of cytopenia did not reveal any significant associations. In patients progressing to sAML, PLK1 expression levels differed significantly according to the presence or absence of resistance to hypomethylation treatment (2,470.58 vs. 415.98, P=0.03). CONCLUSION: PLK1 is upregulated in MDS patients; however, its role in the pathophysiology of MDS is unclear. Gene upregulation in cases with pharmacotherapeutic resistance warrants further investigation.

PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs.

During chondrogenesis from mesenchymal stem cells (MSCs), inadequate differentiation and hypertrophic differentiation are two important limitations. The purpose of this study was to test the hypothesis that chondrogenesis is enhanced and unwanted hypertrophic changes are suppressed by treating bone marrow-derived (BMMSCs) and adipose tissue-derived mesenchymal stem cells (ATMSCs) with parathyroid hormone-related peptide (PTHrP). To induce chondrogenesis, in vitro pellet cultures were carried out using 2.5x10(5) MSCs at passage 3 in chondrogenic medium containing 5 ng/ml of TGF-beta(2) for BMMSCs, and 5 ng/ml of TGF-beta(2) and 100 ng/ml of BMP-7 for ATMSCs. From the 14th day of culture, subsets of pellets were treated with PTHrP [0, 10, 100 ng/ml], and after two more weeks of in vitro culture, pellets were harvested for analysis. The addition of PTHrP dose-dependently increased DNA contents in both BMMSCs and ATMSCs. GAG contents also increased after PTHrP treatment. The gene expression of COL1A1 decreased by three-fourths, while the decrease was not evident in ATMSCs after PTHrP treatment. SOX-9 mRNA increased up to four fold in both BMMSCs and ATMSCs, and COL2A1 gene expression sharply increased to sevenfold in BMMSCs and to 4 fold in ATMSCs. COL10A1 gene expression decreased by a third in both cell types, and Runx-2 expression dropped sharply in both cell types after PTHrP treatment. Safranin-O and immunohistochemistry for type I, II, X collagen and Runx-2 generally paralleled qRT-PCR findings with minor variations. In conclusion, PTHrP was found to promote chondrogenesis and suppress hypertrophy during in vitro chondrogenesis from both BMMSCs and ATMSCs, which supports its use for cartilage tissue engineering.

Epigenetic modification of mesenchymal stromal cells enhances their suppressive effects on the Th17 responses of cells from rheumatoid arthritis patients.

BACKGROUND: The aim of this study was to investigate if epigenetically modified human mesenchymal stromal cells (hMSCs) can regulate the Th17-related immune responses. METHODS: We tested epigenetic drug combinations at various doses and selected the four combinations that resulted in maximal interleukin (IL)-10 and indoleamine 2,3-dioxygenase gene expression in hMSCs. We examined the effects of epigenetically modified hMSCs (epi-hMSCs) on CD4+ T-cell proliferation and inflammatory cytokine secretion under Th0- and Th17-polarizing conditions using mixed lymphocyte reactions and enzyme-linked immunosorbent assays (ELISAs). We determined Th17 cytokine levels and the percentage of Th17 cells among synovial fluid mononuclear cells (SFMCs) from rheumatoid arthritis (RA) patients by ELISA and flow cytometry. RESULTS: Epi-hMSCs inhibited the development of IL-17-producing cells in culture. The percentages of IL-17+ and interferon (IFN)-γ+ cells among peripheral blood mononuclear cells from healthy donors were lower under both the Th0 and Th17 conditions in the presence of epi-hMSCs than in the presence of no or untreated hMSCs. Epi-hMSC-treated RA patient SFMCs secreted lower levels of IL-17 and IFN-γ than RA patient SFMCs cultured without hMSCs or with untreated hMSCs. CONCLUSIONS: An optimal combination of hypomethylating agents and histone deacetylase inhibitors can enhance the immunomodulatory potential of hMSCs, which may be useful for RA treatment.

Enhanced differentiation of mesenchymal stromal cells by three-dimensional culture and azacitidine.

BACKGROUND: Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. METHODS: 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. RESULTS: AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. CONCLUSION: 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.

Effects of mineral trioxide aggregate mixed with hydration accelerators on osteoblastic differentiation.

INTRODUCTION: Despite good physical and biological properties, mineral trioxide aggregate (MTA) has a long setting time. A hydration accelerator could decrease the setting time of MTA. This study assessed the biocompatibility of MTA mixed with hydration accelerators (calcium chloride and low-dose citric acid) and investigated the effect of these materials on osteoblast differentiation. METHODS: Cell viability was evaluated by the EZ-Cytox assay kit (Daeil Lab Service, Seoul, Korea). The gene expressions of osteocalcin and bone sialoprotein were detected by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. The mineralization behavior was evaluated with alizarin red staining. RESULTS: There was no statistically significant difference in cell viability between experimental groups. The messenger RNA level of osteogenic genes significantly increased in MTA mixed with hydration accelerators compared with the control and MTA mixed with water. MTA mixed with the hydration accelerators resulted in similar mineralization compared with MTA mixed with water. CONCLUSIONS: Hydration accelerators increase the osteogenic effect and show a similar effect on the mineralization of MTA, which may have clinical applications.

TrkC promotes survival and growth of leukemia cells through Akt-mTOR-dependent up-regulation of PLK-1 and Twist-1.

It has been suggested that activation of receptor PTKs is important for leukemogenesis and leukemia cell response to targeted therapy in hematological malignancies including leukemia. PTKs induce activation of the PI3K/Akt/mTOR pathway, which can result in prevention of apoptosis. Here, we describe an important role of the TrkC-associated molecular network in the process of leukemogenesis. TrkC was found to be frequently overexpressed in human leukemia cells and leukemia subtypes. In U937 human leukemia cells, blockade of TrkC using small hairpin RNA (shRNA) specific to TrkC or K562a, a specific inhibitor of TrkC, resulted in a significant decrease in growth and survival of the cells, which was closely associated with reduced mTOR level and Akt activity. In addition, TrkC enhances the survival and proliferation of leukemia, which is correlated with activation of the PI3K/Akt pathway. Moreover, TrkC significantly inhibits apoptosis via induction of the expression of PLK-1 and Twist-1 through activation of AKT/mTor pathway; therefore, it plays a key role in leukemogenesis. These findings reveal an unexpected physiological role for TrkC in the pathogenesis of leukemia and have important implications for understanding various hematological malignancies.

Dietary protein restriction induces steatohepatitis and alters leptin/signal transducers and activators of transcription 3 signaling in lactating rats.

Dietary protein restriction during lactation affects lipid metabolism and food intake in rats. The goals of this study were to determine the effect of a low-protein diet on a liver damage in lactating rats, to determine whether dietary protein restriction of lactating dams affects the liver health of their offspring and to elucidate the molecular mechanisms underlying the development of hepatic damage. Lactating Sprague-Dawley rats were fed either a control 20% protein diet or an 8% low-protein diet for 11 or 23 days, respectively. After weaning, the offspring were continuously fed either the same control diet or the low-protein diet for an additional 22 days. Feeding a low-protein diet during lactation caused steatohepatitis with severe steatosis, lobular inflammation, ballooning degeneration and fibrosis. Offspring nourished by dams fed a low-protein diet showed simple hepatic steatosis. Combined effects of increased lipogenesis, decreased fatty acid oxidation and impaired very-low-density lipoprotein secretion were responsible for the development of hepatic steatosis. Hepatic up-regulation of genes linked to oxidative stress including nicotinamide adenine dinucleotide phosphate oxidase, inflammation and fibrogenesis supports the development of steatohepatitis in protein-restricted lactating rats. Furthermore, protein-restricted lactating rats showed activation of the leptin/signal transducers and activators of the transcription 3 signaling pathway. Taken together, oxidative stress induced by up-regulation of nicotinamide adenine dinucleotide phosphate oxidase with activation of leptin/signal transducers and activators of the transcription 3 signaling was responsible for development of steatohepatitis in protein-restricted lactating rats. Our findings suggest that protein malnutrition has a potential to induce steatohepatitis/hepatic steatosis in lactating mothers and infants during breast-feeding.

Electroporation-mediated transfer of SOX trio genes (SOX-5, SOX-6, and SOX-9) to enhance the chondrogenesis of mesenchymal stem cells.

The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-ß1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-ß1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.