Role of Tissue-Specific Stem and Progenitor Cells in the Regeneration of the Pancreas and Testicular Tissue in Diabetic Disorders

Bulletin of Experimental Biology and Medicine - Using the model of hypogonadism in C57Bl/6 male mice, we showed that injection of streptozotocin to newborn animals and high-fat diet induced serum...     

Convenient and Efficient Enrichment of the CD133+ Liver Cells from Rat Fetal Liver Cells as a Source of Liver Stem/Progenitor Cells

Although the stem cells are commonly isolated by FACS or MACS, they are very expensive and these is no specific marker for liver stem/progentior cells (LSPCs). This paper applied a convenient and efficient method to enrich LSPCs. The fetal liver cells (FLCs) were firstly enriched by Percoll discontinuous gradient centrifugation (PDGC) from the rat fetal liver. Then the FLCs in culture were purified to be homogeneous in size by differential trypsinization and differential adherence (DTDA). Flow cytometric analysis revealed more than half of the purified FLCs expressed alternative markers of LSPCs (CD117, c-Met, Sca-1, CD90, CD49f and CD133). In other words, the purified FLCs were heterogeneous. Therefore, they were sequentially layered into six fractions by Percoll continuous gradient centrifugation (PCGC). Both CD133 and CD49f expressed decreasingly from fraction 1 to 6. In fraction 1 and 2, about 85% FLCs expressed CD133, which were revealed to be LSPCs by high expressions of AFP and CK-19, low expressions of G-6-P and ALB. To conclude, the purity of CD133⁺ LSPCs enriched by combination of PDGC, DTDA and PCGC is close to that obtained by MACS. This study will greatly contribute to two important biological aspects: liver stem cells isolation and liver cell therapy.     

Modulation of Reparative Regeneration and CD117 Expression by Liver Cells after Partial Hepatectomy in Mice

The effects of 3-aminophthalhydrazide and carrageenan on reparative regeneration and expression of CD117 by liver cells after partial hepatectomy were studied in mice. 3-Aminophthalhydrazide stimulated regeneration of the liver and increased the count of CD117⁺ hepatocytes. By contrast, carrageenan inhibited liver reparation and CD117 expression.     

Stem Cells in Liver Regeneration and Their Potential Clinical Applications

Stem cells constitute a population of “primitive cells” with the ability to divide indefinitely and give rise to specialized cells under special conditions. Because of these two characteristics they have received particular attention in recent decades. These cells are the primarily responsible factors for the regeneration of tissues and organs and for the healing of lesions, a feature that makes them a central key in the development of cell-based medicine, called Regenerative Medicine. The idea of wound and organ repair and body regeneration is as old as the mankind, reflecting the human desire for inhibiting aging and immortality and it is first described in the ancient Greek myth of Prometheus. It is of interest that the myth refers to liver, an organ with remarkable regenerative ability after loss of mass and function caused by liver injury or surgical resection. Over the last decade there has been an important progress in understanding liver physiology and the mechanisms underlying hepatic development and regeneration. As liver transplantation, despite its difficulties, remains the only effective therapy for advanced liver disease so far, scientific interest has nowadays been orientated towards Regenerative Medicine and the use of stem cells to repair damaged liver. This review is focused on the available literature concerning the role of stem cells in liver regeneration. It summarizes the results of studies concerning endogenous liver regeneration and stem cell experimental protocols. Moreover, this review discusses the clinical studies that have been conducted in humans so far.     

Role of BMP Signaling in Pancreatic Progenitor Differentiation from Human Embryonic Stem Cells

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.     

Beta-Catenin Activation Promotes Liver Regeneration after Acetaminophen-Induced Injury

Acute liver failure (ALF) remains a disease with poor patient outcome. Improved prognosis is associated with spontaneous liver regeneration, which supports the relevance of exploring ‘regenerative’ therapies. Therefore, the role of the Wnt/β-catenin pathway in liver regeneration following ALF was investigated. ALF was induced in mice by acetaminophen overdose, which is also a leading cause of liver failure in patients. β-catenin distribution was also studied in liver sections from acetaminophen-induced ALF patients. A nonlethal dose of acetaminophen, which induces liver regeneration, led to stabilization and activation of β-catenin for 1 to 12 hours. These data were also verified by increased expression of the β-catenin surrogate target glutamine synthetase. β-Catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3β and an increase in levels of casein kinase 2α, and led to increased cyclin-D1, another known β-catenin target. These observations were next substantiated in β-catenin conditional-null mice (β-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in β-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in β-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear β-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of β-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.Acute liver failure (ALF) is an ominous liver disease that is on the rise in the Western world.^1,2 The current treatments for ALF include an infusion of N-acetyl cysteine, a glutathione precursor, or orthotopic liver transplantation (OLT). While N-acetyl cysteine has been used with success in a subset of ALF patients secondary to acetaminophen (APAP) overdose, its effectiveness in non-APAP-associated ALF cases is being debated.^3,4 OLT remains a mainstay treatment in severe cases and is associated with high cost, morbidity and mortality; and is limited by available donor livers. With over 50% of ALF cases being due to overdose of the commonly used over-the-counter anti-pyretic and analgesic APAP, efforts are under to way to investigate significance of regenerative therapies in ALF patients.^5,6,7 Recently, studies have demonstrated that patients who have a timely increase in spontaneous liver regeneration following APAP overdose have improved prognosis.^8,9 The role of liver regeneration in protection against APAP-induced ALF has also been demonstrated in experimental models.Liver possesses a remarkable self-regenerative capability, as has been shown extensively in rodents after partial hepatectomy or by toxicant-induced injury with carbon tetrachloride, chloroform and thioacetamide.^{10,11,12,13,14} Regeneration is mediated by intricate signaling via growth factors such as hepatocyte growth factor and epidermal growth factor, and cytokines such as tumor necrosis factor-α. Recent studies have demonstrated a critical role of the Wnt/β-catenin pathway in hepatocyte proliferation during liver development, cancer, and regeneration.¹⁵ Rapid activation of β-catenin in rat liver was observed within 5 minutes of partial hepatectomy and knockdown of β-catenin with antisense treatment resulted decreased liver regeneration.^16,17 In addition, lack of β-catenin in mice results in delayed liver regeneration due to inadequate G1 to S transition.^18,19 These studies also identified CYP2E1 and CYP1A2 to be dramatically lower in β-catenin-null livers, thus precluding the use of these mice to test regeneration after APAP-injury. CYP2E1 and CYP1A2 are essential to metabolize APAP to induce injury and regeneration.²⁰The present study was designed to investigate changes in the Wnt/β-catenin pathway during liver regeneration in ALF induced by APAP. We identified β-catenin activation early during this event, which appears to be important in stimulating regeneration in this model. In addition, we showed high correlation between nuclear/cytoplasmic β-catenin and ongoing regeneration in hepatic biopsies from ALF patients. Our data suggest β-catenin activation as one of the mechanisms that may contribute to spontaneous regeneration following ALF in preclinical and clinical scenario.     

Role of Bile Acids in Liver Injury and Regeneration following Acetaminophen Overdose

Bile acids play a critical role in liver injury and regeneration, but their role in acetaminophen (APAP)–induced liver injury is not known. We tested the effect of bile acid modulation on APAP hepatotoxicity using C57BL/6 mice, which were fed a normal diet, a 2% cholestyramine (CSA)–containing diet for bile acid depletion, or a 0.2% cholic acid (CA)–containing diet for 1 week before treatment with 400 mg/kg APAP. CSA-mediated bile acid depletion resulted in significantly higher liver injury and delayed regeneration after APAP treatment. In contrast, 0.2% CA supplementation in the diet resulted in a moderate delay in progression of liver injury and significantly higher liver regeneration after APAP treatment. Either CSA-mediated bile acid depletion or CA supplementation did not affect hepatic CYP2E1 levels or glutathione depletion after APAP treatment. CSA-fed mice exhibited significantly higher activation of c-Jun N-terminal protein kinases and a significant decrease in intestinal fibroblast growth factor 15 mRNA after APAP treatment. In contrast, mice fed a 0.2% CA diet had significantly lower c-Jun N-terminal protein kinase activation and 12-fold higher fibroblast growth factor 15 mRNA in the intestines. Liver regeneration after APAP treatment was significantly faster in CA diet–fed mice after APAP administration secondary to rapid cyclin D1 induction. Taken together, these data indicate that bile acids play a critical role in both initiation and recovery of APAP-induced liver injury.Bile acids are versatile biological molecules that regulate energy homeostasis, activate nuclear receptors and cell signaling pathways, and control cell proliferation and inflammatory processes in the liver and gastrointestinal tract.^1,2 Bile acids maintain their own homeostasis by activating a complex signaling network involving hepatic and intestinal farnesoid X receptor (FXR), small heterodimer partner, and intestinal fibroblast growth factor (FGF) 15 (FGF19 in human) expression, culminating in inhibition of the primary bile acid–synthesizing enzyme, CYP7A1.^3–6 Although bile acids are potent signaling molecules at pathophysiological concentrations, they cause apoptosis, necrosis, and oxidative stress.^3,7–10 Bile acids have also been implicated in stimulation of liver regeneration.^11–14 Studies in recent years indicate that the bile acid–mediated gut-liver signaling axis may play a critical role in regulation of liver homeostasis.^6,15,16Acetaminophen (APAP) is the most commonly used analgesic and antipyretic agent.¹⁷ An overdose of APAP is the major cause of acute liver failure in the United States.^18,19 The mechanisms of APAP-induced liver injury and subsequent liver regeneration are the focus of intense investigation.^20–22 In an overdose situation, excess APAP is mainly metabolized by CYP2E1 to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). In hepatocytes, conjugation of NAPQI to GSH is the key mechanism for detoxification of NAPQI. Once the GSH is depleted, NAPQI attacks cellular proteins, especially mitochondrial proteins, to form protein adducts. This triggers a cascade of intracellular signaling events involving c-Jun N-terminal protein kinase (JNK) activation and mitochondrial permeability transition, finally culminating in necrotic cell death.²⁰ Liver injury is followed by compensatory liver regeneration, which is a critical determinant of final outcome of liver injury.²³ Despite decades of research, how these intracellular events are affected by extracellular signaling is not known.The current study was designed to explore the role of bile acids in initiation of liver injury and stimulation of liver regeneration after APAP overdose. These studies are highly significant because the data reveal a novel role of bile acids in cellular protection and liver regeneration after APAP overdose, and these studies investigate the effect of resin-mediated bile acid depletion, a commonly used therapy, on APAP toxicity.     

The Role of Liver Sinusoidal Cells in Hepatocyte-Directed Gene Transfer

Hepatocytes are a key target for gene therapy of inborn errors of metabolism as well as of acquired diseases such as liver cancer and hepatitis. Gene transfer efficiency into hepatocytes is significantly determined by histological and functional aspects of liver sinusoidal cells. On the one hand, uptake of vectors by Kupffer cells and liver sinusoidal endothelial cells may limit hepatocyte transduction. On the other hand, the presence of fenestrae in liver sinusoidal endothelial cells provides direct access to the space of Disse and allows vectors to bind to receptors on the microvillous surface of hepatocytes. Nevertheless, the diameter of fenestrae may restrict the passage of vectors according to their size. On the basis of lege artis measurements of the diameter of fenestrae in different species, we show that the diameter of fenestrae affects the distribution of transgene DNA between sinusoidal and parenchymal liver cells after adenoviral transfer. The small diameter of fenestrae in humans may underlie low efficiency of adenoviral transfer into hepatocytes in men. The disappearance of the unique morphological features of liver sinusoidal endothelial cells in pathological conditions like liver cirrhosis and liver cancer may further affect gene transfer efficiency. Preclinical gene transfer studies should consider species differences in the structure and function of liver sinusoidal cells as important determinants of gene transfer efficiency into hepatocytes.The liver is a central organ in many metabolic processes. Numerous inherited metabolic disorders have their origin in the liver. Therefore, hepatocytes are a key target for gene transfer directed at correction of inborn errors of metabolism and of hemophilia. Inborn errors of metabolism may lead to accumulation of toxic products in hepatocytes and extensive hepatotoxicity, as observed in disorders like α₁-antitrypsin deficiency, type I tyrosinemia, or Wilson disease. In other metabolic diseases, such as in Crigler-Najjar syndrome type I, ornithine transcarbamylase deficiency, familial hypercholesterolemia, and hemophilia A and B, manifestations are primarily extrahepatic. In addition, the liver is a target for gene therapy of acquired diseases such as liver cancer and hepatitis.Insights into the determinants of gene transfer efficiency to hepatocytes are therefore required to evaluate the potential of gene therapy for inborn errors of metabolism and for acquired liver diseases. These determinants include innate and adaptive immune responses, cellular and biochemical determinants of hepatocyte transduction such as ligand receptor interactions, and anatomical and histological factors. In this minireview, we focus predominantly on the role of liver sinusoidal cells and sinusoidal fenestrae as determinants of the efficiency in hepatocyte-directed gene transfer.     

Investigation of the Role of Glypican 3 in Liver Regeneration and Hepatocyte Proliferation

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans.¹ Six glypicans have been identified in mammals so far (GPC1 to GPC6) and two members of this family have been found in Drosophila (dally and dlp).^2,3 Although the homology of amino acids between glypican members is moderate, all glypicans are approximately 60 to 70 kd in size and share a characteristic pattern of 14 conserved cysteine residues.⁴ Intact glypicans are decorated with heparan sulfate (HS), which is located in the last 50 amino acids of the C terminus, placing the HS chains close to the cell membrane.⁵GPC3 is located on the X chromosome, and is highly expressed during embryogenesis and organogenesis.^2,6 In the adult, on the other hand, GPC3 can only be detected in a limited number of tissues, including the lung, ovaries, mammary epithelium, and mesothelium.^2,7 GPC3 is highly up-regulated in hepatocellular carcinoma, one of the most common solid malignancies in the world that accounts for about 1 million deaths each year.⁸ Our previous study and others have shown that GPC3 is highly up-regulated in hepatocellular carcinoma and hepatoblastoma, but not in normal liver or tissue adjacent to tumors.^8,9 The soluble form of GPC3 was also identified in the serum of patients with hepatocellular carcinomas, and can be used as a serological test for the diagnosis of hepatocellular carcinoma.^10,11 GPC3 is also involved in cell proliferation in some hepatoma cell lines.¹²The stage- and tissue-specific pattern of expression suggests that GPC3 is involved in morphogenesis and development. It is reported that a loss-of -function mutation in the GPC3 gene causes Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal overgrowth, increased risk of embryonic tumors during early childhood, and numerous visceral and skeletal anomalies.^13,14 The involvement of GPC3 in Simpson-Golabi-Behmel syndrome was confirmed by the generation of GPC3-deficient mice (GPC3−/−), since these mice display some of the phenotypic features of Simpson-Golabi-Behmel syndrome, including developmental overgrowth (∼30%), general enlargement of multiple organ including liver, respiratory infections, cystic kidneys, etc.¹⁵ These findings suggest that GPC3 plays a negative growth regulatory role despite its over-expression in liver cancer.Since loss of function in GPC3 in Simpson-Golabi-Behmel syndrome leads to overgrowth of many organs including liver in humans and mice, it is reasonable to speculate that GPC3 functions as a growth inhibitor in the liver. Given the overexpression of GPC3 in human liver cancer, we wanted to study the role of GPC3 in hepatocyte growth regulation. To that end, we used the rodent model of liver regeneration after partial hepatectomy (PHx) in which hepatocyte growth dynamics are well characterized.^16,17 We also used hepatocyte primary cultures, in which we and others have characterized the hepatocyte growth cycle under the influence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF).¹⁸ In liver regeneration after 2/3 PHx,¹⁹ specific rat liver lobes are removed intact (about 2/3 of the total mass) without damage to the lobes left behind. The residual lobes enlarge to make up for the mass of the removed lobes, though the resected lobes never grow back.¹⁹ Following liver PHx, hepatocytes enter into the cell cycle from their habitual quiescent phase and proliferate to restore normal hepatic mass and hepatic functional capacity. The whole process lasts 5 to 7 days in rats, during which the hepatocytes divide first at about 24 hours post-PHx, followed by the biliary ductular cells, then the Kupffer cells and stellate cells, and finally the endothelial cells.¹⁷ Details of this process and the dynamics of proliferation of different cell types, role of growth factors and cytokines have been described.¹⁷Our results show that GPC3 protein and RNA levels are increased in the later stages of liver regeneration, as well as in the later stages of growth cessation in HGF and EGF stimulated hepatocyte cultures. When GPC3 protein is knocked down using Morpholino oligonucleotides (oligos), hepatocyte growth is promoted at the end of proliferation. Thus we hypothesize that GPC3 could negatively regulate liver regeneration and hepatocyte proliferation.To investigate the potential proteins that could interact with GPC3, a yeast two-hybrid assay was performed, and several interesting proteins, including CD81, were found to interact with GPC3. CD81, also called TAPA-1 (target of an antiproliferative antibody) is a widely expressed cell surface tetraspanin involved in an astonishing variety of biological responses.²⁰ It has been cloned independently several times for different functional effects and is reported to influence adhesion, morphology, activation, proliferation, and differentiation of B, T, and other cells.^21,22 CD81 is also reported to interact with HCV glycoprotein E2 and assist with HCV infection in hepatocytes.²³ In our research, CD81 RNA and protein levels changed in the same manner as GPC3. In addition, co-immunofluorescence and co-immunoprecipitation were performed that verified the interaction and localization of GPC3 and CD81 in rat liver, suggesting that CD81 interaction is likely to form an important relationship with ligands to GPC3 function.     

人动员外周血成体干/祖细胞定向诱导分化为血管内皮前体细胞的免疫表型分析

观察动员后的人外周血成体干/祖细胞体外定向诱导分化为血管内皮前体细胞（EPC）的免疫表型特征及变化。采用健康成人经粒细胞集落刺激因子（rhG-CSF）动员后的外周血成体干/祖细胞,经贴壁培养法定向分化为EPC,之后通过流式细胞术检测EPC表型。结果显示,培养后的贴壁细胞具有内皮细胞特征,能够与I型荆豆凝集素（UEA-I）结合。流式细胞术检测UEA-I结合率为92.1%,内皮细胞标志物CD31、KDR、CD62E均有不同程度增加,而粒-巨噬细胞集落生成单位（CFU-GM）及CD34表达明显减低,表明诱导分化后细胞的免疫表型发生了相应的变化。     

The Role of Innate Immune Cells in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a very common hepatic pathology featuring steatosis and is linked to obesity and related conditions, such as the metabolic syndrome. When hepatic steatosis is accompanied by inflammation, the disorder is defined as nonalcoholic steatohepatitis (NASH), which in turn can progress toward fibrosis development that can ultimately result in cirrhosis. Cells of innate immunity, such as neutrophils or macrophages, are central regulators of NASH-related inflammation. Recent studies utilizing new experimental technologies, such as single-cell RNA sequencing, have revealed substantial heterogeneity within the macrophage populations of the liver, suggesting distinct functions of liver-resident Kupffer cells and recruited monocyte-derived macrophages with regards to regulation of liver inflammation and progression of NASH pathogenesis. Herein, we discuss recent developments concerning the function of innate immune cell subsets in NAFLD and NASH.     

Mesenchymal-Epithelial Transition in Culture of Stromal Progenitor Cells Isolated from the Liver of a Patient with Alcoholic Cirrhosis

Bulletin of Experimental Biology and Medicine - The cells isolated from biopsy specimen of a patient with alcoholic liver cirrhosis and cultured under standard conditions for obtaining stromal cell...     

肝再生相关细胞因子的探究

肝脏再生是一个极其复杂、多因素参与的过程,而其中的细胞因子起着至关重要的作用。这些因子主要包括:起始因子或者前炎性因子如肿瘤坏死因子(TNFα)、白细胞介素6(IL6)等;肝细胞生长因子(HGF)等生长因子;辅助肝细胞分裂原如胰岛素和肾上腺素等;转化生长因子β(TGFβ)等持续性肝细胞生长抑制因子;生长抑制因子的抑制因子,如:LAP(TGFβN末端肽段)等。前三类细胞因子分别对处在G0期、G1期、S期的肝细胞起直接或间接作用,而后两类因子对前三种因子起到促进或者抑制的作用。本文旨在探讨肝再生各主要相关细胞因子在肝再生过程中的作用机制以及它们之间的相互关系。     

Ultrastructural Studies of Glioma Stem Cells/Progenitor Cells

Although the ultrastructural features of several brain tumor cells have been studied in details, the ultrastructure of glioma stem cells/progenitors cells (GSPC) has rarely been reported. In this paper, the authors describe the ultrastructural features of GSPCs isolated from both a glioma tissue and the human glioma cell SHG-44 cell line. The ultrastructural features of the two kinds of GSPCs were similar, with relatively developed mitochondria, Golgi apparatuses, ribosomes, undeveloped rough endoplasmic reticula, seldom lysosomes and no typical autophagosomes, and high nuclear–cytoplasmic ratio. Their nuclei, frequently containing huge amounts of euchromatin and a small quantity of heterochromatin, were mostly globular; and the majority of the nuclei had only one nucleole. Typical apoptotic cells could hardly be found in tumor spheres, and between adjacent cells there were cell junctions, which probably were incompletely developed desmosomes or intermediate junctions. In conclusion, their ultrastructural features showed that GSPCs were at the primary stage of differentiation, and could even partially reveal the underlying reasons for the malignant proliferation and differential inhibition of GSPCs.