Jinfeng Yang

Therapeutic potential of transplanted placental mesenchymal stem cells in treating Chinese miniature pigs with acute liver failure.

BackgroundStem cell-based therapy to treat liver diseases is a focus of current research worldwide. So far, most such studies depend on rodent hepatic failure models. The purpose of this study was to isolate mesenchymal stem cells from human placenta (hPMSCs) and determine their therapeutic potential for treating Chinese experimental miniature pigs with acute liver failure (ALF).MethodshPMSCs were isolated and analyzed for their purity and differentiation potential before being employed as the donor cells for transplantation. ALF models of Chinese experimental miniature pigs were established and divided into four groups: no cell transplantation; hPMSCs transplantation via the jugular vein; X-ray-treated hPMSCs transplantation via the portal vein; and hPMSCs transplantation via the portal vein. The restoration of biological functions of the livers receiving transplantation was assessed via a variety of approaches such as mortality rate determination, serum biochemical analysis, and histological, immunohistochemical, and genetic analysis.ResultshPMSCs expressed high levels of CD29, CD73, CD13, and CD90, had adipogenic, osteogenic, and hepatic differentiation potential. They improved liver functions in vivo after transplantation into the D-galactosamine-injured pig livers as evidenced by the fact that ALT, AST, ALP, CHE, TBIL, and TBA concentrations returned to normal levels in recipient ALF pigs. Meanwhile, histological data revealed that transplantation of hPMSCs via the portal vein reduced liver inflammation, decreased hepatic denaturation and necrosis, and promoted liver regeneration. These ameliorations were not found in the other three groups. The result of 7-day survival rates suggested that hPMSCs transplantation via the portal vein was able to significantly prolong the survival of ALF pigs compared with the other three groups. Histochemistry and RT-PCR results confirmed the presence of transplanted human cells in recipient pig livers (Groups III, IV).ConclusionsOur data revealed that hPMSCs could not only differentiate into hepatocyte-like cells in vitro and in vivo, but could also prolong the survival time of ALF pigs. Regarding the transplantation pathways, the left branch of the portal vein inside the liver was superior to the jugular vein pathway. Thus, hPMSCs transplantation through the portal vein by B-ultrasonography may represent a superior approach for treating liver diseases.

In vivo hepatic differentiation of mesenchymal stem cells from human umbilical cord blood after transplantation into mice with liver injury.

AIMnThe aim of this study was to analyze the hepatic differentiation potential of human umbilical cord blood-derived mesenchymal stem cells (hUCBMSCs) after transplantation into severe combined immune deficiency (SCID) mice with liver injury induced by D-galactosamine/lipopolysaccharide (GalN/LPS) and to explore the possibility that cells can partially repair GalN/LPS-induced hepatic damage.nnnMETHODSnMononuclear cells (MNCs) were isolated from fresh human umbilical cord blood, characterized by flow cytometry, and then transplanted into GalN/LPS-injured mice. Specimens were collected at 7, 14, 21, and 28 days after hUCBMSC transplantation. Histopathological changes were analyzed by hematoxylin and eosin staining. Polymerase chain reaction (PCR) for a specific marker of human cells, the human Alu sequence, was performed to locate exogenous hUCBMSCs in mouse livers. Expression of human hepatocyte-specific markers such as human albumin (hALB), human alpha-fetoprotein (hAFP), human cytokeratin 18 (hCK18), and human cytokeratin 19 (hCK19) were analyzed by reverse transcriptase (RT)-PCR and immunohistochemical staining.nnnRESULTSnThe hUCBMSCs were positive for the human MSC-specific markers CD271, CD29, CD90, CD105, and CD73, but negative for CD31, CD79b, CD133, CD34, and CD45. Histological findings showed that the hepatic damage in mice was attenuated after hMSC administration, and liver architecture was much better preserved. Human cells in the injured liver of recipient mice were detected by PCR for the human Alu sequence. In addition, expression of markers of hepatic lineage, including hALB, hAFP, hCK18, and hCK19, was detected by immunohistochemistry and RT-PCR in mouse livers after hUCBMSC transplantation, suggesting the formation of hepatocyte-like cells in vivo.nnnCONCLUSIONnMSCs from hUCB exhibit the potential to differentiate into hepatocyte-like cells in the livers of hUCB-transplanted mice as well as partially repair the liver damage induced by GalN/LPS.

Proteomic analysis of regenerating mouse liver following 50% partial hepatectomy

BackgroundAlthough 70% (or 2/3) partial hepatectomy (PH) is the most studied model for liver regeneration, the hepatic protein expression profile associated with lower volume liver resection (such as 50% PH) has not yet been reported. Therefore, the aim of this study was to determine the global protein expression profile of the regenerating mouse liver following 50% PH by differential proteomics, and thereby gaining some insights into the hepatic regeneration mechanism(s) under this milder but clinically more relevant condition.ResultsProteins from sham-operated mouse livers and livers regenerating for 24 h after 50% PH were separated by SDS-PAGE and analyzed by nanoUPLC-Q-Tof mass spectrometry. Compared to sham-operated group, there were totally 87 differentially expressed proteins (with 50 up-regulated and 37 down-regulated ones) identified in the regenerating mouse livers, most of which have not been previously related to liver regeneration. Remarkably, over 25 differentially expressed proteins were located at mitochondria. Several of the mitochondria-resident proteins which play important roles in citric acid cycle, oxidative phosphorylation and ATP production were found to be down-regulated, consistent with the recently-proposed model in which the reduction of ATP content in the remnant liver gives rise to early stress signals that contribute to the onset of liver regeneration. Pathway analysis revealed a central role of c-Myc in the regulation of liver regeneration.ConclusionsOur study provides novel evidence for mitochondria as a pivotal organelle that is connected to liver regeneration, and lays the foundation for further studies on key factors and pathways involved in liver regeneration following 50% PH, a condition frequently used for partial liver transplantation and conservative liver resection.

GFP Labeling and Hepatic Differentiation Potential of Human Placenta-Derived Mesenchymal Stem Cells

Background: Stem cell-based therapy in liver diseases has received increasing interest over the past decade, but direct evidence of the homing and implantation of transplanted cells is conflicting. Reliable labeling and tracking techniques are essential but lacking. The purpose of this study was to establish human placenta-derived mesenchymal stem cells (hPMSCs) expressing green fluorescent protein (GFP) and to assay their hepatic functional differentiation in vitro. Methods: The GFP gene was transduced into hPMSCs using a lentivirus to establish GFP+ hPMSCs. GFP+ hPMSCs were analyzed for their phenotypic profile, viability and adipogenic, osteogenic and hepatic differentiation. The derived GFP+ hepatocyte-like cells were evaluated for their metabolic, synthetic and secretory functions, respectively. Results: GFP+ hPMSCs expressed high levels of HLA I, CD13, CD105, CD73, CD90, CD44 and CD29, but were negative for HLA II, CD45, CD31, CD34, CD133, CD271 and CD79. They possessed adipogenic, osteogenic and hepatic differentiation potential. Hepatocyte-like cells derived from GFP+ hPMSCs showed typical hepatic phenotypes. Conclusions: GFP gene transduction has no adverse influences on the cellular or biochemical properties of hPMSCs or markers. GFP gene transduction using lentiviral vectors is a reliable labeling and tracking method. GFP+ hPMSCs can therefore serve as a tool to investigate the mechanisms of MSC-based therapy, including hepatic disease therapy.

Hypoxia-inducible factor-2 alpha promotes the proliferation of human placenta-derived mesenchymal stem cells through the MAPK/ERK signaling pathway.

Human placenta-derived mesenchymal stem cells (hPMSCs) reside in a physiologically low-oxygen microenvironment. Hypoxia influences a variety of stem cell cellular activities, frequently involving hypoxia-inducible factor-2 alpha (HIF-2α). This research showed that hPMSCs cultured in hypoxic conditions (5% O2) exhibited a more naïve morphology and had a higher proliferative capability and higher HIF-2α expression than hPMSCs cultured in normoxic conditions (21% O2). Similar to the hypoxic cultures, hPMSCs over-expressing HIF-2α showed higher proliferative potential and higher expression of CCND1 (CyclinD1), MYC (c-Myc), POU5F1 (Oct4) and the components of the MAPK/ERK pathway. In contrast, these genes were down-regulated in the HIF-2α-silenced hPMSCs. After adding the MAPK/ERK inhibitor PD0325901, cell growth and the expression of CCND1 and MYC were inhibited. Furthermore, the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) showed that HIF-2α bound to the MAPK3 (ERK1) promoter, indicative of its direct regulation of MAPK/ERK components at the transcriptional level during hPMSC expansion. Taken together, our results suggest that HIF-2α facilitated the preservation of hPMSC stemness and promoted their proliferation by regulating CCND1 and MYC through the MAPK/ERK signaling pathway.

Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice.

BACKGROUNDnCell therapy has been promising for various diseases. We investigated whether transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) has any therapeutic effects on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced fulminant hepatic failure in mice.nnnMETHODSnhUCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deficiency mice with GalN/LPS-induced fulminant hepatic failure. After transplantation, the localization and differentiation of hUCMSCs in the injured livers were investigated by immunohistochemical and genetic analyses. The recovery of the injured livers was evaluated histologically. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test.nnnRESULTSnhUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adipogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for human alu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the formation of hUCMSCs-derived hepatocyte-like cells in vivo.nnnCONCLUSIONSnhUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUCMSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

A metabonomics study of Chinese miniature pigs with acute liver failure treated with transplantation of placental mesenchymal stem cells

Metabonomics has become a highly sensitive and powerful tool for screening of biomarkers and elucidation of biochemical processes. Recently, we reported beneficial therapeutic effect of human placenta mesenchymal stem cells (hPMSCs) transplantation on d-galactosamine (GalN)-induced liver injury in Chinese miniature pigs. However, the underlying mechanism remains largely unclear. Here, UPLC/TOF/MS-based metabonomics approach was employed to analyze serum from GalN-treated pigs with the goal of identifying potential biomarkers for acute liver injury. Our results showed that obvious metabolic disturbance occurred during acute liver failure (ALF), which can be ameliorated by the hPMSCs transplantation. The metabolic profiles of the hPMSCs transplantation group returned back to the original state 5xa0weeks after the hPMSCs transplantation. In addition, the result obtained from metabolic trajectory analysis correlated well with those from biochemical analysis and histological examination. Serum levels of several metabolites including glycoursodeoxycholic acid, glycochenodeoxycholic acid, aromatic amino acids, phosphatidylcholine, lysophosphatidylcholine and sphingomyelin, were significantly modified during the process of ALF and cell treatment. Taken together, our study has gained new insight into the molecular mechanism on how hPMSCs administration facilitates the recovery of ALF, and provided strong support for treating liver diseases with stem cell-based therapies.

Homology modeling and molecular dynamics simulation of the HIF2α degradation-related HIF2α-VHL complex.

BACKGROUNDnHypoxia-inducible factor 2 alpha (HIF2α), prolyl hydroxylase domain protein 2 (PHD2), and the von Hippel Lindau tumor suppressor protein (pVHL) are three principal proteins in the oxygen-sensing pathway. Under normoxic conditions, a conserved proline in HIF2α is hydroxylated by PHD2 in an oxygen-dependent manner, and then pVHL binds and promotes the degradation of HIF2α. However, the crystal structure of the HIF2α-pVHL complex has not yet been established, and this has limited research on the interaction between HIF and pVHL. Here, we constructed a structural model of a 23-residue HIF2α peptide (528-550)-pVHL-ElonginB-ElonginC complex by using homology modeling and molecular dynamics simulations. We also applied these methods to HIF2α mutants (HYP531PRO, F540L, A530u2009V, A530T, and G537R) to reveal structural defects that explain how these mutations weaken the interaction with pVHL.nnnMETHODSnHomology modeling and molecular dynamics simulations were used to construct a three-dimensional (3D) structural model of the HIF2α-VHL complex. Subsequently, MolProbity, an active validation tool, was used to analyze the reliability of the model. Molecular mechanics energies combined with the generalized Born and surface area continuum solvation (MM-GBSA) and solvated interaction energy (SIE) methods were used to calculate the binding free energy between HIF2a and pVHL, and the stability of the simulation system was evaluated by using root mean square deviation (RMSD) analysis. We also determined the secondary structure of the system by using the definition of secondary structure of proteins (DSSP) algorithm. Finally, we investigated the structural significance of specific point mutations known to have clinical implications.nnnRESULTSnWe established a reliable structural model of the HIF2α-pVHL complex, which is similar to the crystal structure of HIF1α in 1LQB. Furthermore, we compared the structural model of the HIF2α-pVHL complex and the HIF2α (HYP531P, F540L, A530V, A530T, and G537R)-pVHL mutants on the basis of RMSD, DSSP, binding free energy, and hydrogen bonding. The experimental data indicate that the stability of the structural model of the HIF2α-pVHL complex is higher than that of the mutants, consistently with clinical observations.nnnCONCLUSIONSnThe structural model of the HIF2α-pVHL complex presented in this study enhances understanding of how HIF2α is captured by pVHL. Moreover, the important contact amino acids that we identified may be useful in the development of drugs to treat HIF2a-related diseases.

Impact of Oxygen Concentration on Metabolic Profile of Human Placenta-Derived Mesenchymal Stem Cells As Determined by Chemical Isotope Labeling LC–MS

The placenta resides in a physiologically low oxygen microenvironment of the body. Hypoxia induces a wide range of stem cell cellular activities. Here, we report a workflow for exploring the role of physiological (hypoxic, 5% oxygen) and original cell culture (normoxic, 21% oxygen) oxygen concentrations in regulating the metabolic status of human placenta-derived mesenchymal stem cells (hPMSCs). The general biological characteristics of hPMSCs were assessed via a variety of approaches such as cell counts, flow cytometry and differentiation study. A sensitive 13C/12C-dansyl labeling liquid chromatography-mass spectrometry (LC-MS) method targeting the amine/phenol submetabolome was used for metabolic profiling of the cell and corresponding culture supernatant. Multivariate and univariate statistical analyses were used to analyze the metabolomics data. hPMSCs cultured in hypoxia display smaller size, higher proliferation, greater differentiation ability and no difference in immunophenotype. Overall, 2987 and 2860 peak pairs or metabolites were detected and quantified in hPMSCs and culture supernatant, respectively. Approximately 86.0% of cellular metabolites and 84.3% of culture supernatant peak pairs were identified using a dansyl standard library or matched to metabolite structures using accurate mass search against human metabolome libraries. The orthogonal partial least-squares discriminant analysis (OPLS-DA) showed a clear separation between the hypoxic group and the normoxic group. Ten metabolites from cells and six metabolites from culture supernatant were identified as potential biomarkers of hypoxia. This study demonstrated that chemical isotope labeling LC-MS can be used to reveal the role of oxygen in the regulation of hPMSC metabolism, whereby physiological oxygen concentrations may promote arginine and proline metabolism, pantothenate and coenzyme A (CoA) biosynthesis, and alanine, aspartate and glutamate metabolism.

Metabolic profiling associated with autophagy of human placenta-derived mesenchymal stem cells by chemical isotope labeling LC−MS

Abstract Autophagy has been reported to have a pivotal role in maintaining stemness, regulating immunomodulation and enhancing the survival of mesenchymal stem cells (MSCs). However, the effect of autophagy on MSC metabolism is largely unknown. Here, we report a workflow for examining the impact of autophagy on human placenta‐derived MSC (hPMSC) metabolome profiling with chemical isotope labeling (CIL) LC−MS. Rapamycin or 3‐methyladenine was successfully used to induce or inhibit autophagy, respectively. Then, 12C‐ and 13C‐dansylation labeling LC−MS were used to profile the amine/phenol submetabolome. A total of 935 peak pairs were detected and 50 metabolites were positively identified using the dansylation metabolite standards library, and 669 metabolites were putatively identified based on an accurate mass match in metabolome databases. 12C/13C‐p‐dimethylaminophenacyl bromide labeling LC−MS was used to analyze the carboxylic acid submetabolome; 4736 peak pairs were detected, among which 33 metabolites were positively identified in the dimethylaminophenacyl metabolite standards library, and 3007 metabolites were putatively identified. PCA/OPLS‐DA analysis combined with volcano plots and Venn diagrams was used to determine the significant metabolites. Metabolites pathway analysis demonstrated that hPMSCs appeared to generate more ornithine with the arginine and proline metabolism pathway and utilized more pantothenic acid to synthesize acetyl‐CoA in the beta‐alanine metabolism pathway when autophagy was activated. Meanwhile, acetyl‐CoA conversion to fatty acids led to accumulation in the fatty acid biosynthesis pathway. In contrast, when autophagy was suppressed, a reduction in metabolites demonstrated weakened metabolic activity in these metabolic pathways. Our research provides a more comprehensive understanding of hPMSC metabolism associated with autophagy.