Aim To purify hepatocyte regeneration stimulatory factor from shark liver and research its molecular feature and activity. Methods and Results Hepatocyte regeneration stimulatory factor (sHRSF) was isolated from hea...Aim To purify hepatocyte regeneration stimulatory factor from shark liver and research its molecular feature and activity. Methods and Results Hepatocyte regeneration stimulatory factor (sHRSF) was isolated from healthy shark livers and separated by homogenization, freezing melting, heat treating, centrifugation, and ultrafiltration. HRSF activity was found mainly in the subfraction of molecular weight less than 30 000 daltons. This crude ultrafiltrate was further purified successively by DEAE Sepharose fast flow chromatography, FPLC Resource 30Q, Resource Q and Mono Q chromatography. A single band was displayed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to molecular weight of 14 600 daltons. The characteristic absorption was obtained at the wavelength 276 nm. The isoelectric point was about 5 1. It contained 18 amino acids and the 15 N terminal amino acid residues were LVGPIGAVGPAGKDG. It had a significant activity in stimulating liver to regenerate. Conclusion We obtained an unknown new active protein, that is hepatocyte regeneration stimulatory factor from shark liver (sHRSF).展开更多
Megakaryocytes and hepatocytes are unique cells in mammals that undergo polyploidization through endomitosis in terminal differentiation.Many polyploidization regulators and underlying mechanisms have been reported,mo...Megakaryocytes and hepatocytes are unique cells in mammals that undergo polyploidization through endomitosis in terminal differentiation.Many polyploidization regulators and underlying mechanisms have been reported,most of which are tightly coupled with development,organogenesis,and cell differentiation.However,the nature of endomitosis,which involves successful entry into and exit from mitosis without complete cytokinesis,has not yet been fully elucidated.We highlight that endomitosis is a new cell fate in the cell cycle,and tetraploidy is a critical stage at the bifurcation of cell fate decision.This review summarizes the recent research progress in this area and provides novel insights into how cells manipulate mitosis toward endomitosis.Endomitotic cells can evade the tetraploidy restrictions and proceed to multiple rounds of the cell cycle.This knowledge not only deepens our understanding of endomitosis as a fundamental biological process but also offers new perspectives on the physiological and pathophysiological implications of polyploidization.展开更多
The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-pro-duced hepatocytes.By performing serial alloge...The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-pro-duced hepatocytes.By performing serial allogeneic hepatocyte transplantation in CRISPR/Cas9-mediated Fah-knockout pigs,we successfully achieved large-scale ex-pansion of hepatocytes while maintaining their authentic biological characteristics.Particularly,the established model enables sustained in vivo liver reconstruction,concurrently ameliorating hepatic fibrosis and demonstrating functional microenvi-ronmental remodeling.Moreover,through comprehensive single-cell transcriptomic profiling of 52418 hepatocytes across transplant generations(F0-F2),we discovered that the cellular composition of these transplanted hepatocytes is similar to that of wild-type hepatocytes.The regenerated liver exhibits all six major hepatic cell types identical to the wild-type counterparts,with the characteristic lobular zonation pat-terns well preserved.Our research provides valuable insights into the large-scale expansion of physiologically functional hepatocytes in vivo without compromising their biological properties.This finding holds great promise for advancing the clinical application of human hepatocyte transplantation,potentially offering more effective treatment options for patients with liver diseases.展开更多
Non-alcoholic fatty liver disease(NAFLD)has become the most prevalent chronic liver disease globally,with its incidence rising annually.It can progress to cirrhosis and even hepatocellular carcinoma,posing a serious t...Non-alcoholic fatty liver disease(NAFLD)has become the most prevalent chronic liver disease globally,with its incidence rising annually.It can progress to cirrhosis and even hepatocellular carcinoma,posing a serious threat to human health.Stress can participate in the pathological process of NAFLD by activating inflammatory responses and regulating levels of inflammatory mediators,with hepatocyte injury being a core component of NAFLD progression.This paper focuses on three key stress-related inflammatory mediators:tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and C-reactive protein(CRP),elucidating their core mechanisms in the pathway related to stress signal,followed by inflammatory activation and hepatocyte injury respectively,and reviewing current research.Research indicates that certain inflammatory mediators can damage hepatocytes by directly inducing apoptosis or indirectly regulating metabolic disorders and fibrosis progression.However,questions regarding causal relationships,target specificity for intervention,and quantification of psychological stress remain unresolved.This paper aims to provide theoretical support for NAFLD intervention strategies targeting inflammatory mediators,clarifying future research directions to advance clinical translation.展开更多
Metabolic dysfunction-associated steatotic liver disease,characterized by pathological intracellular triglyceride(TG)accumulation,is mechanistically associated with the disrupted spatiotemporal regulation of hepatocyt...Metabolic dysfunction-associated steatotic liver disease,characterized by pathological intracellular triglyceride(TG)accumulation,is mechanistically associated with the disrupted spatiotemporal regulation of hepatocyte nuclear factor(HNF)-dependent transcriptional programs.HNFs,including key members such as HNF-1α,HNF-4α,and HNF-6,constitute a liver-enriched family of transcription factors that govern hepatic lipid metabolism through hierarchical transcriptional regulatory networks.These networks critically regulate the dynamic equilibrium of TG metabolism,encompassing TG synthesis,storage,lipolysis,and lipoprotein-mediated export.This review comprehensively deciphers the molecular cascades through which HNF dysfunction exacerbates TG metabolic disorder in metabolic dysfunction-associated steatotic liver disease.Additionally,we evaluate emerging translational strategies targeting key HNF regulatory nodes and discuss current clinical challenges as well as potential solutions.展开更多
BACKGROUND Bletilla striata polysaccharides(BSP)have antioxidant,immune regulation,and anti-fibrotic activities.However,the therapeutic effect and mechanisms underlying the action of BSP in metabolic dysfunction-assoc...BACKGROUND Bletilla striata polysaccharides(BSP)have antioxidant,immune regulation,and anti-fibrotic activities.However,the therapeutic effect and mechanisms underlying the action of BSP in metabolic dysfunction-associated steatotic liver disease(MASLD)have not been fully understood.AIMTo investigate the therapeutic effects and mechanisms of BSP on MASLD by centering on the hepatocyte nuclearfactor kappa B p65(RelA)/hepatocyte nuclear factor-1 alpha(HNF1α)signaling.METHODSA mouse model of MASLD was induced by feeding with a high-fat-diet(HFD)and a hepatocyte model of steatosiswas induced by treatment with sodium oleate(SO)and sodium palmitate(SP).The therapeutic effects of BSP onMASLD were examined in vivo and in vitro.The mechanisms underlying the action of BSP were analyzed for theireffect on lipid metabolism disorder,endoplasmic reticulum(ER)stress,and the RelA/HNF1αsignaling.RESULTSHFD feeding reduced hepatocyte RelA and HNF1αexpression,induced ER stress,lipid metabolism disorder,andnecroptosis in mice,which were significantly mitigated by treatment with BSP.Furthermore,treatment with BSP orBSP-containing conditional rat serum significantly attenuated the sodium oleate/sodium palmitate(SO/SP)-induced hepatocyte steatosis by decreasing lipid accumulation,and lipid peroxidation,and enhancing theexpression of RelA,and HNF1α.The therapeutic effects of BSP on MASLD were partially abrogated by RELAsilencing in mice and RELA knockout in hepatocytes.RELA silencing or knockout significantly down-regulatedHNF1αexpression,and remodeled ER stress and oxidative stress responses during hepatic steatosis.CONCLUSIONTreatment with BSP ameliorates MASLD,associated with enhancing the RelA/HNF1αsignaling,remodeling ERstress and oxidative stress responses in hepatocytes.展开更多
BACKGROUND Glucotoxic pancreaticβcells impair glycogenesis of hepatocytes,with exosomes serving as novel mediators.miR-375-3p is the most abundant miRNA in the pancreas and critical forβ-cell function,but whether it...BACKGROUND Glucotoxic pancreaticβcells impair glycogenesis of hepatocytes,with exosomes serving as novel mediators.miR-375-3p is the most abundant miRNA in the pancreas and critical forβ-cell function,but whether it plays a role in pancreasliver crosstalk remains unclear.AIM To investigate the role of miR-375-3p,a key regulator of pancreaticβcells,in remotely regulating hepatocyte glycogenesis via exosomes.METHODS Mice fed a high-fat diet(HFD)served as animal models,and mouse primary pancreatic islet cells and theβ-cell line MIN-6 were used as cellular models.miR-375-3p expression in pancreatic cells,hepatocytes and exosomes was detected in both animal and cellular models.Transwell assays,exosome treatment,and exosome-depleted supernatant culture were used to investigate the role of exosomal miR-375-3p in pancreatic-hepatocyte crosstalk.The AKT/GSK signaling pathway and hepatic glycogen content were used as indicators to evaluate hepatocyte glycogenesis.Luciferase reporter assays were used to evaluate the downstream targets of miR-375-3p.RESULTS Increased levels of miR-375-3p were observed in both the pancreas and liver of HFD-fed mice.In contrast to the in vivo results,high-glucose treatment exclusively increased the expression of miR-375-3p in pancreatic cells but had no effect on hepatocytes.Furthermore,hepatocytes treated with the supernatant and exosomes from glucotoxic pancreatic cells presented elevated expression of miR-375-3p.Additionally,exosomal transfer of miR-375-3p from pancreatic cells to hepatocytes suppressed the AKT/GSK signaling pathway,thereby reducing the hepatic glycogen content.Luciferase analysis indicated that the recombination signal binding protein for the immunoglobulin kappa J region(Rbpj)is a target gene of miR-375-3p.Rbpj inhibition impaired hepatic glycogenesis,and Rbpj overexpression reversed the effect on glycogenesis induced by miR-375-3p.CONCLUSION Pancreatic cell-derived miR-375-3p can be delivered to hepatocytes via exosomes and inhibits hepatocyte glycogenesis by targeting Rbpj.展开更多
The natural estrogens,including estrone(E_(1)),17β-estradiol(E_(2)),and estriol(E_(3)),are frequently detected in aquatic environment at relatively high levels.The most commonly used biomarkers for estrogens are main...The natural estrogens,including estrone(E_(1)),17β-estradiol(E_(2)),and estriol(E_(3)),are frequently detected in aquatic environment at relatively high levels.The most commonly used biomarkers for estrogens are mainly expressed in the liver of fish.Analyses of the global gene profiling in fish liver cells under estrogens treatment will provide precise toxicogenomic information of the natural estrogens which is still not well known.In this study,we developed methods for isolation and culture of primary hepatocytes from liver tissue of male Chinese medaka(Oryzias sinensis),and analyzed the global gene expression profiling in the primary hepatocytes treated with E_(1)(1,10,and 100 nmol/L),E_(2)(0.01,0.1,1,10,and 100 nmol/L),and E_(3)(1,10,and 100 nmol/L)using RNA-seq.It was found that 175,248,and 218 genes were differentially expressed in the E_(1),E_(2),and E_(3) groups,respectively.These differentially expressed genes(DEGs)were mainly enriched in Gene Ontology(GO)terms of“response to estradiol”,“response to estrogen”,and“lipid transport”.Of the DEGs,vitellogenin genes,including vtg1,vtg2,and vtg3,were the mostly up-regulated and followed by zona pellucida genes which include zp2.3,zp2l1 and zp3a.2.In addition,genes of slc41a1,zp2.1,esr1,pkd1l1,fam^(2)0ca,best3,etc.were also obviously up-regulated by the estrogens in concentration-dependent patterns.RT-qPCR was used to validate the results of RNA-seq and found that vtg2 should be the best biomarker gene for estrogen study,which could well response to natural estrogens and weak estrogenic chemical,propyl 4-hydroxybenzoate.展开更多
AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.
This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation(HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue f...This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation(HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that:(1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver;(2) Organs with steatosis(≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained;(3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT;(4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and(5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a veryuseful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.展开更多
AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcin...AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.展开更多
AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepat...AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.展开更多
AIM: To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH).
AIM： To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable pro...AIM： To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation. METHODS： Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells. Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development. RESULTS： Mean viable hepatocyte yield was 9.3×10^6 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 10^6 hepatocytes, representing a maximum tumor purging efficacy of greater than 400000-fold. Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells. CONCLUSION： Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis. Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.展开更多
AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expr...AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.展开更多
AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-...AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-containing cytokine mixture of interleukin-1β,tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors,e.g.hepatocyte growth factor,epidermal growth factor and/or transforming growth factor-α.Cells were analyzed for glutathione levels.Culture supernatants were assayed for produc-tion of reactive oxygen intermediates(ROIs) as well as NO2-,NO3-and S-nitrosothiols.To determine cellular damage,release of aspartate aminotransferase(AST) into the culture medium was analyzed.Activation of nuclear factor(NF)-κB was measured by electrophoretic mobility shift assay.RESULTS:Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation.AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture,independent of growth-factor co-stimulation.However,pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione.Application of growth factors did not result in increased NF-κB activation.Pretreatment with growth factors further increased formation of NO2-,NO3-and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture.Thus,we propose that,together with an increase in glutathione increased NO2-,NO3-formation might shift their metabolism towards non-toxic products.CONCLUSION:Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.展开更多
We present 2 cases of hepatocyte nuclear factor 1α (HNF1α)-mutated adenomatosis, discovered for reasons unrelated to this disease, and identified using immunohistochemical methods. These new tools may further our un...We present 2 cases of hepatocyte nuclear factor 1α (HNF1α)-mutated adenomatosis, discovered for reasons unrelated to this disease, and identified using immunohistochemical methods. These new tools may further our understanding of the link between adenomas/adenomatosis subtypes and their complications, and their association with other abnormalities.展开更多
AIM： TO analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid （4n） and octoploid （Sn） nuclei, and then compared mononuclear and binuclear hepatocyte populations： METHODS： T...AIM： TO analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid （4n） and octoploid （Sn） nuclei, and then compared mononuclear and binuclear hepatocyte populations： METHODS： The percentages of mononuclear diploid （2n）, 4n, and 8n hepatocytes and those of binuclear 2 × 2n, 2 × 4n, and 2 × 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62 patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 × 4n and 2 × 8n hepatocytes in the binuclear hepatocyte population. RESULTS： The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 ×4n and 2 × 8n hepatocytes in binuclear hepatocytes were similar, regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus. CONCLUSION： The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.展开更多
Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inheritedliver diseases and liver failure.It is a relatively less complicated surgical procedure,and has the advantage...Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inheritedliver diseases and liver failure.It is a relatively less complicated surgical procedure,and has the advantage that it can be repeated several times if unsuccessful.Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation.Despite these advantages hepatocyte transplantation is less popular.Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes.Generation of "hepatocyte like cells"/i Heps from embryonic stem cells(ES) and induced pluripotent stem cells(iP SCs) by directed differentiation is an emerging solution to the latter issue.Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is,being explored.However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or i PSC.There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells".Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol.Directed differentiation protocols need to be designed,compared,analyzed and tweaked systematically and logically than empirically.There is a need for a wellcoordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.展开更多
基金NationalMarine863Project (No .2 0 0 1AA62 40 90),NationalNaturalScienceFoundationofChina (No .3 0 17110 3 )
文摘Aim To purify hepatocyte regeneration stimulatory factor from shark liver and research its molecular feature and activity. Methods and Results Hepatocyte regeneration stimulatory factor (sHRSF) was isolated from healthy shark livers and separated by homogenization, freezing melting, heat treating, centrifugation, and ultrafiltration. HRSF activity was found mainly in the subfraction of molecular weight less than 30 000 daltons. This crude ultrafiltrate was further purified successively by DEAE Sepharose fast flow chromatography, FPLC Resource 30Q, Resource Q and Mono Q chromatography. A single band was displayed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to molecular weight of 14 600 daltons. The characteristic absorption was obtained at the wavelength 276 nm. The isoelectric point was about 5 1. It contained 18 amino acids and the 15 N terminal amino acid residues were LVGPIGAVGPAGKDG. It had a significant activity in stimulating liver to regenerate. Conclusion We obtained an unknown new active protein, that is hepatocyte regeneration stimulatory factor from shark liver (sHRSF).
基金supported by the National Natural Science Foundation of China(Nos.32270643,91957109,and 81870427).
文摘Megakaryocytes and hepatocytes are unique cells in mammals that undergo polyploidization through endomitosis in terminal differentiation.Many polyploidization regulators and underlying mechanisms have been reported,most of which are tightly coupled with development,organogenesis,and cell differentiation.However,the nature of endomitosis,which involves successful entry into and exit from mitosis without complete cytokinesis,has not yet been fully elucidated.We highlight that endomitosis is a new cell fate in the cell cycle,and tetraploidy is a critical stage at the bifurcation of cell fate decision.This review summarizes the recent research progress in this area and provides novel insights into how cells manipulate mitosis toward endomitosis.Endomitotic cells can evade the tetraploidy restrictions and proceed to multiple rounds of the cell cycle.This knowledge not only deepens our understanding of endomitosis as a fundamental biological process but also offers new perspectives on the physiological and pathophysiological implications of polyploidization.
基金National Key Research and Development Program of China,Grant/Award Number:2021YFA0805905,2023YFC3404305 and 2024YFA1107900the Strategic Priority Research Program of the Chinese Academy of Sciences,Grant/Award Number:XDB1150000+1 种基金the CAS Project for Young Scientists in Basic Research,Grant/Award Number:YSBR-012Bingtuan Science and Technology Project,Grant/Award Number:NYHXGG2023AA01。
文摘The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-pro-duced hepatocytes.By performing serial allogeneic hepatocyte transplantation in CRISPR/Cas9-mediated Fah-knockout pigs,we successfully achieved large-scale ex-pansion of hepatocytes while maintaining their authentic biological characteristics.Particularly,the established model enables sustained in vivo liver reconstruction,concurrently ameliorating hepatic fibrosis and demonstrating functional microenvi-ronmental remodeling.Moreover,through comprehensive single-cell transcriptomic profiling of 52418 hepatocytes across transplant generations(F0-F2),we discovered that the cellular composition of these transplanted hepatocytes is similar to that of wild-type hepatocytes.The regenerated liver exhibits all six major hepatic cell types identical to the wild-type counterparts,with the characteristic lobular zonation pat-terns well preserved.Our research provides valuable insights into the large-scale expansion of physiologically functional hepatocytes in vivo without compromising their biological properties.This finding holds great promise for advancing the clinical application of human hepatocyte transplantation,potentially offering more effective treatment options for patients with liver diseases.
基金Science Research Project of Hebei Education Department(Project No.:QN2022013)。
文摘Non-alcoholic fatty liver disease(NAFLD)has become the most prevalent chronic liver disease globally,with its incidence rising annually.It can progress to cirrhosis and even hepatocellular carcinoma,posing a serious threat to human health.Stress can participate in the pathological process of NAFLD by activating inflammatory responses and regulating levels of inflammatory mediators,with hepatocyte injury being a core component of NAFLD progression.This paper focuses on three key stress-related inflammatory mediators:tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and C-reactive protein(CRP),elucidating their core mechanisms in the pathway related to stress signal,followed by inflammatory activation and hepatocyte injury respectively,and reviewing current research.Research indicates that certain inflammatory mediators can damage hepatocytes by directly inducing apoptosis or indirectly regulating metabolic disorders and fibrosis progression.However,questions regarding causal relationships,target specificity for intervention,and quantification of psychological stress remain unresolved.This paper aims to provide theoretical support for NAFLD intervention strategies targeting inflammatory mediators,clarifying future research directions to advance clinical translation.
基金Supported by the Science and Technology Planning Projects of Guizhou Province,No.QKHJC-MS[2025]384the Science and Technology Planning Projects of Zunyi City,No.ZSKHHZ(2023)470+3 种基金the WBE Liver Fibrosis Foundation,No.CFHPC2025028Chinese Foundation for Hepatitis Prevention and Control Muxin Research Fund of Chronic Hepatitis B,No.MX202404Beijing Liver and Gallbladder Mutual Aid Public Welfare Foundation Artificial Liver Special Fund,No.iGandanF-1082024-RGG018the Student Innovation and Entrepreneurship Training Program of Zunyi Medical University,No.2024106610923.
文摘Metabolic dysfunction-associated steatotic liver disease,characterized by pathological intracellular triglyceride(TG)accumulation,is mechanistically associated with the disrupted spatiotemporal regulation of hepatocyte nuclear factor(HNF)-dependent transcriptional programs.HNFs,including key members such as HNF-1α,HNF-4α,and HNF-6,constitute a liver-enriched family of transcription factors that govern hepatic lipid metabolism through hierarchical transcriptional regulatory networks.These networks critically regulate the dynamic equilibrium of TG metabolism,encompassing TG synthesis,storage,lipolysis,and lipoprotein-mediated export.This review comprehensively deciphers the molecular cascades through which HNF dysfunction exacerbates TG metabolic disorder in metabolic dysfunction-associated steatotic liver disease.Additionally,we evaluate emerging translational strategies targeting key HNF regulatory nodes and discuss current clinical challenges as well as potential solutions.
基金National Natural Science Foundation of China,No.32260089Science and Technology Research Foundation of Guizhou Province,No.QKHJC-ZK(2022)YB642+3 种基金Science and Technology Research Foundation of Hubei Province,No.2022BCE030Science and Technology Research Foundation of Changzhou City,No.CE20225040Science and Technology Research Foundation of Zunyi City,No.ZSKHHZ(2022)344 and No.ZSKHHZ(2022)360WBE Liver Fibrosis Foundation,No.CFHPC2025028.
文摘BACKGROUND Bletilla striata polysaccharides(BSP)have antioxidant,immune regulation,and anti-fibrotic activities.However,the therapeutic effect and mechanisms underlying the action of BSP in metabolic dysfunction-associated steatotic liver disease(MASLD)have not been fully understood.AIMTo investigate the therapeutic effects and mechanisms of BSP on MASLD by centering on the hepatocyte nuclearfactor kappa B p65(RelA)/hepatocyte nuclear factor-1 alpha(HNF1α)signaling.METHODSA mouse model of MASLD was induced by feeding with a high-fat-diet(HFD)and a hepatocyte model of steatosiswas induced by treatment with sodium oleate(SO)and sodium palmitate(SP).The therapeutic effects of BSP onMASLD were examined in vivo and in vitro.The mechanisms underlying the action of BSP were analyzed for theireffect on lipid metabolism disorder,endoplasmic reticulum(ER)stress,and the RelA/HNF1αsignaling.RESULTSHFD feeding reduced hepatocyte RelA and HNF1αexpression,induced ER stress,lipid metabolism disorder,andnecroptosis in mice,which were significantly mitigated by treatment with BSP.Furthermore,treatment with BSP orBSP-containing conditional rat serum significantly attenuated the sodium oleate/sodium palmitate(SO/SP)-induced hepatocyte steatosis by decreasing lipid accumulation,and lipid peroxidation,and enhancing theexpression of RelA,and HNF1α.The therapeutic effects of BSP on MASLD were partially abrogated by RELAsilencing in mice and RELA knockout in hepatocytes.RELA silencing or knockout significantly down-regulatedHNF1αexpression,and remodeled ER stress and oxidative stress responses during hepatic steatosis.CONCLUSIONTreatment with BSP ameliorates MASLD,associated with enhancing the RelA/HNF1αsignaling,remodeling ERstress and oxidative stress responses in hepatocytes.
基金Supported by Beijing Natural Science Foundation,No.7252124National High Level Hospital Clinical Research Funding,No.BJ-2024-219,No.BJ-2025-125 and No.BJ-2023-236+1 种基金National Natural Science Foundation of China,No.82370584,No.82470395 and No.81600618National Key R&D Program of China,No.2021YFE0114200.
文摘BACKGROUND Glucotoxic pancreaticβcells impair glycogenesis of hepatocytes,with exosomes serving as novel mediators.miR-375-3p is the most abundant miRNA in the pancreas and critical forβ-cell function,but whether it plays a role in pancreasliver crosstalk remains unclear.AIM To investigate the role of miR-375-3p,a key regulator of pancreaticβcells,in remotely regulating hepatocyte glycogenesis via exosomes.METHODS Mice fed a high-fat diet(HFD)served as animal models,and mouse primary pancreatic islet cells and theβ-cell line MIN-6 were used as cellular models.miR-375-3p expression in pancreatic cells,hepatocytes and exosomes was detected in both animal and cellular models.Transwell assays,exosome treatment,and exosome-depleted supernatant culture were used to investigate the role of exosomal miR-375-3p in pancreatic-hepatocyte crosstalk.The AKT/GSK signaling pathway and hepatic glycogen content were used as indicators to evaluate hepatocyte glycogenesis.Luciferase reporter assays were used to evaluate the downstream targets of miR-375-3p.RESULTS Increased levels of miR-375-3p were observed in both the pancreas and liver of HFD-fed mice.In contrast to the in vivo results,high-glucose treatment exclusively increased the expression of miR-375-3p in pancreatic cells but had no effect on hepatocytes.Furthermore,hepatocytes treated with the supernatant and exosomes from glucotoxic pancreatic cells presented elevated expression of miR-375-3p.Additionally,exosomal transfer of miR-375-3p from pancreatic cells to hepatocytes suppressed the AKT/GSK signaling pathway,thereby reducing the hepatic glycogen content.Luciferase analysis indicated that the recombination signal binding protein for the immunoglobulin kappa J region(Rbpj)is a target gene of miR-375-3p.Rbpj inhibition impaired hepatic glycogenesis,and Rbpj overexpression reversed the effect on glycogenesis induced by miR-375-3p.CONCLUSION Pancreatic cell-derived miR-375-3p can be delivered to hepatocytes via exosomes and inhibits hepatocyte glycogenesis by targeting Rbpj.
基金supported by the Fisheries Innovation Team of Beijing Agriculture Innovation Consortium(No.BAIC07–2023–02)the National Natural Science Foundation of China(Nos.22176004 and 21777003).
文摘The natural estrogens,including estrone(E_(1)),17β-estradiol(E_(2)),and estriol(E_(3)),are frequently detected in aquatic environment at relatively high levels.The most commonly used biomarkers for estrogens are mainly expressed in the liver of fish.Analyses of the global gene profiling in fish liver cells under estrogens treatment will provide precise toxicogenomic information of the natural estrogens which is still not well known.In this study,we developed methods for isolation and culture of primary hepatocytes from liver tissue of male Chinese medaka(Oryzias sinensis),and analyzed the global gene expression profiling in the primary hepatocytes treated with E_(1)(1,10,and 100 nmol/L),E_(2)(0.01,0.1,1,10,and 100 nmol/L),and E_(3)(1,10,and 100 nmol/L)using RNA-seq.It was found that 175,248,and 218 genes were differentially expressed in the E_(1),E_(2),and E_(3) groups,respectively.These differentially expressed genes(DEGs)were mainly enriched in Gene Ontology(GO)terms of“response to estradiol”,“response to estrogen”,and“lipid transport”.Of the DEGs,vitellogenin genes,including vtg1,vtg2,and vtg3,were the mostly up-regulated and followed by zona pellucida genes which include zp2.3,zp2l1 and zp3a.2.In addition,genes of slc41a1,zp2.1,esr1,pkd1l1,fam^(2)0ca,best3,etc.were also obviously up-regulated by the estrogens in concentration-dependent patterns.RT-qPCR was used to validate the results of RNA-seq and found that vtg2 should be the best biomarker gene for estrogen study,which could well response to natural estrogens and weak estrogenic chemical,propyl 4-hydroxybenzoate.
基金Supported by Major Scientific and Technological Project of Shandong Province,No.201221019Cisco Clinical Oncology Research Fund and Bayer Schering Cancer Research Fund,No.Y-B2012-011
文摘AIM: To establish a method for the reversible immortalization of human hepatocytes, which may offer a good and safe source of hepatocytes for practical applications.
文摘This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation(HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that:(1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver;(2) Organs with steatosis(≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained;(3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT;(4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and(5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a veryuseful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.
基金Supported by The Major Scientific and Technological Project of Hubei Province, No. 2007ABD005
文摘AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination. RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes. CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.
基金Supported by the National Natural Science Foundation of China,No.30772129Jiangsu Provincial Key Medical Center for Hepatobiliary Disease,No.ZX200605
文摘AIM： To investigate whether the function of hepatocytes co-cultured with bone marrow mesenchymal stem cells （MSCs） could be maintained in serum from acute-on- chronic liver failure （ACLF） patients.METHODS： Hepatocyte supportive functions and cy- totoxicity of sera from 18 patients with viral hepatitis B-induced ACLF and 18 healthy volunteers were evalu- ated for porcine hepatocytes co-cultured with MSCs and hepatocyte mono-layered culture, respectively. Chemo- kine profile was also examined for the normal serum and liver failure serum.RESULTS： Hepatocyte growth factor （HGF） and Tumor necrosis factor; tumor necrosis factor （TNF）-a were re- markably elevated in response to ACLF while epidermal growth factor （EGF） and VEGF levels were significantly decreased. Liver failure serum samples induced a higher detachment rate, lower viability and decreased liver sup- port functions in the homo-hepatocyte culture. Hepato-cytes co-cultured with MSCs could tolerate the cytotoxic- ity of the serum from ACLF patients and had similar liver support functions compared with the hepatocytes cul- tured with healthy human serum in vitro. In addition, co- cultured hepatocytes maintained a proliferative capability despite of the insult from liver failure serum.CONCLUSION： ACLF serum does not impair the cell morphology, viability, proliferation and overall metabolic capacities of hepatocyte co-cultured with MSCs in vitro.
文摘AIM: To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric non-alcoholic steatohepatitis (NASH).
文摘AIM： To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation. METHODS： Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells. Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development. RESULTS： Mean viable hepatocyte yield was 9.3×10^6 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 10^6 hepatocytes, representing a maximum tumor purging efficacy of greater than 400000-fold. Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells. CONCLUSION： Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis. Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.
基金Supported by the "Matthias Lackas-Stiftung", "Paul und Ursula Klein-Stiftung", "Heinrich und Erna Schaufler-Stiftung", "Gisela Stadelmann-Stiftung", and study grants from the Johann Wolfgang Goethe-Universitatsklinikum,Universitatsklinikum Essen (IFORES),and Deutsche Forschungsgemeinschaft (AU 117/4-1)
文摘AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.
基金Supported by The Federal Ministry of Research (BMBF-01 GN0984)
文摘AIM:To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis.METHODS:Rat hepatocytes,isolated by collagenase perfusion,were incubated with a lipopolysaccharide(LPS)-containing cytokine mixture of interleukin-1β,tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors,e.g.hepatocyte growth factor,epidermal growth factor and/or transforming growth factor-α.Cells were analyzed for glutathione levels.Culture supernatants were assayed for produc-tion of reactive oxygen intermediates(ROIs) as well as NO2-,NO3-and S-nitrosothiols.To determine cellular damage,release of aspartate aminotransferase(AST) into the culture medium was analyzed.Activation of nuclear factor(NF)-κB was measured by electrophoretic mobility shift assay.RESULTS:Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation.AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture,independent of growth-factor co-stimulation.However,pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione.Application of growth factors did not result in increased NF-κB activation.Pretreatment with growth factors further increased formation of NO2-,NO3-and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture.Thus,we propose that,together with an increase in glutathione increased NO2-,NO3-formation might shift their metabolism towards non-toxic products.CONCLUSION:Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.
文摘We present 2 cases of hepatocyte nuclear factor 1α (HNF1α)-mutated adenomatosis, discovered for reasons unrelated to this disease, and identified using immunohistochemical methods. These new tools may further our understanding of the link between adenomas/adenomatosis subtypes and their complications, and their association with other abnormalities.
文摘AIM： TO analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid （4n） and octoploid （Sn） nuclei, and then compared mononuclear and binuclear hepatocyte populations： METHODS： The percentages of mononuclear diploid （2n）, 4n, and 8n hepatocytes and those of binuclear 2 × 2n, 2 × 4n, and 2 × 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62 patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 × 4n and 2 × 8n hepatocytes in the binuclear hepatocyte population. RESULTS： The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 ×4n and 2 × 8n hepatocytes in binuclear hepatocytes were similar, regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus. CONCLUSION： The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.
基金Supported by IIP fellowship(2013-2014)Albert Einstein College of Medicine,New York,through the generosity of the Gruss Lipper Family Foundation
文摘Hepatocyte transplantation is an alternative to liver transplantation in certain disorders such as inheritedliver diseases and liver failure.It is a relatively less complicated surgical procedure,and has the advantage that it can be repeated several times if unsuccessful.Another advantage is that hepatocytes can be isolated from partly damaged livers which are not suitable for liver transplantation.Despite these advantages hepatocyte transplantation is less popular.Important issues are poor engraftment of the transplanted cells and the scarcity of donor hepatocytes.Generation of "hepatocyte like cells"/i Heps from embryonic stem cells(ES) and induced pluripotent stem cells(iP SCs) by directed differentiation is an emerging solution to the latter issue.Direct conversation or trans-differentiation of fibroblasts to "hepatocyte like cells" is another way which is,being explored.However this method has several inherent and technical disadvantages compared to the directed differentiation from ES or i PSC.There are several methods claiming to be "highly efficient" for generating "highly functional" "hepatocyte like cells".Currently different groups are working independently and coming up with differentiation protocols and each group claiming an advantage for their protocol.Directed differentiation protocols need to be designed,compared,analyzed and tweaked systematically and logically than empirically.There is a need for a wellcoordinated global initiative comparable to the Human Genome Project to achieve this goal in the near future.
