Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting

BACKGROUND/AIMS: Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. METHODS: Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. RESULTS: When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. CONCLUSIONS: Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.     

Salivary gland progenitor cells induced by duct ligation differentiate into hepatic and pancreatic lineages

Tissue damage can be assessed based on regenerative responses, including progenitor cell proliferation. In the salivary gland, tissue damage induced by ligation of main ducts leads to the disappearance of acinar cells and to marked proliferation of ductal cells. Reopening of the ducts leads to repopulation of acinar cells within 1 to 2 weeks, which suggests activation of tissue progenitor cells in a damaged state. Because submandibular glands derive from the endoderm and ectoderm, we investigated the possibility of the presence of endodermal progenitor cells. We cultured cells obtained from the ligated salivary gland and identified colonies of epithelium-like cells. We singled out and purified the cells by limited dilution, and one of the cells designated SGP-1 was used for further experiments. The SGP-1 expresses both alpha6beta1 integrin and cytoplasmic laminin. The hematopoietic stem cell marker CD34 and hepatic oval cell markers such as albumin, alpha-fetoprotein (AFP), and cytokeratin 19 are all negative. However, when SGP-1 cells were transplanted into the liver via the portal vein, these cells were integrated into hepatic trabecula and produced albumin. When SGP-1 cells formed clusters on type I collagen-coated dishes, they differentiated into endodermal lineage and 2 major types of clusters appeared: one contained cells positive for AFP and/or albumin (hepatic cluster) and the other positive for glucagon and/or insulin (pancreatic cluster). On laminin-coated dishes, SGP-1 selectively differentiated into hepatic-type cells. In conclusion, the multipotent progenitor cells isolated from the rat salivary gland have characteristics of tissue stem cells and can differentiate into cells of endodermal lineages.     

Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells

BACKGROUND AND OBJECTIVES: The aim of this study was to determine whether Hoechst effluxing side population cells isolated from murine liver represent hepatic stem cells, and to examine whether hepatic side population cells arise from bone marrow side population cells. DESIGN AND METHODS: Side population cells were isolated from murine liver by flow cytometry after Hoechst staining and injected directly into murine livers of animals pre-treated with the hepatotoxin 3,5 diethoxy carbonyl-1, 4-dihydrocollidine (DDC). Y-chromosome in situ hybridization was used to track donor cells in the livers. In addition, bone marrow side population cells were stably engrafted into the hematopoietic system of sublethally irradiated recipients and CD45 alleleic staining and Y-chromosome in situ hybridization were used to track side population cell progeny in the liver. RESULTS: In vitro, CD45pos and CD45neg hepatic SP cells gave rise to hematopoietic colonies and mixed colonies of hematopoietic and hepatic differentiation. After orthotopic liver cell transplantation, donor hepatic side population cells contributed to the regeneration of mature liver parenchyma and bile duct epithelium. After transplantation of bone marrow side population cells, both CD45pos and CD45neg hepatic side population cells were partially derived from donor stem cells and could be recruited to repair liver damage after treatment with DDC. INTERPRETATION AND CONCLUSIONS: These findings introduce hepatic side population cells as a facultative liver-regenerating population, reveal interchangeability of tissue stem cells at the level of the side population, and suggest that bone marrow-derived side population cells might be exploited for the repair of diseased or damaged liver.     

Characterization of hepatic progenitors from human fetal liver using CD34 as a hepatic progenitor marker

AIM: To enrich putative hepatic progenitors from the developing human fetal liver using CD34 as a marker. METHODS: Aborted fetuses of 13-20 wk were used for the isolation of liver cells. The cells were labeled with anti CD34; a marker used for isolating progenitor population and the cells were sorted using magnetic cell sorting. The positive fractions of cells were assessed for specific hepatic markers. Further, these cells were cultured in vitro for long term investigation. RESULTS: Flow cytometric and immunocytochemical analysis for alphafetoprotein (AFP) showed that the majority of the enriched CD34 positive cells were positive for AFP. Furthermore, these enriched cells proliferated in the long term and maintained hepatic characteristics in in vitro culture. CONCLUSION: The study shows that aborted human fetal liver is a potential source for isolation of hepatic progenitors for clinical applications. The study also demonstrates that CD34 can be a good marker for the enrichment of progenitor populations.     

In vitro evidence for the presence of hematopoietic stem cells in the adult human liver

Previous studies have identified novel lymphoid phenotypes in the adult human liver and provided evidence to suggest that lymphoid differentiation can occur locally in this organ. The aim of this study was to examine the adult human liver for the presence of hematopoietic stem cells that may provide the necessary precursor population for local hematopoietic and lymphoid differentiation. Hepatic mononuclear cells (HMNC) were extracted from normal adult liver biopsy specimens using a combination of mechanical disruption and enzymatic digestion. The stem cell marker CD34 was found on 0.81% to 2.35% of isolated HMNCs by flow cytometry. CD34(+) HMNCs were positively selected using magnetically labeled beads, and the enriched population was further examined for surface markers characteristically expressed by immature hematopoietic cells and early progenitors. CD45 was expressed by 49% (+/-23%) of CD34(+) HMNCs, indicating their hematopoietic origin. CD38, one of the first markers to be expressed by developing progenitor cells was found on 50% (+/-22%) of CD34(+) HMNCs indicating the presence of both pluripotent stem cells and committed precursors. The majority (90%) of CD34(+) HMNCs coexpressed the activation marker human leukocyte antigen DR, consistent with actively cycling cells. Functional maturation of these hepatic progenitors was shown by the detection of multilineage hematopoietic colony formation after tissue culture. Erythroid (BFU-E), granulocyte-monocyte (CFU-GM), and mixed colonies (CFU-GEMM) were detected after culture of unseparated HMNCs and the enriched CD34(+) HMNC population; 14.3 +/- 13.2 (mean +/- SD) BFU-E, 3.1 +/- 3.1 CFU-GM, and 0.4 +/- 0.9 CFU-GEMM per 1 x 10(5) unseparated HMNCs and 16.0 +/- 9.5 BFU-E and 1.7 +/- 0.9 CFU-GM were identified per 2.4 x 10(3) CD34(+) HMNCs plated. The detection of surface markers characteristic of immature hematopoietic cells and colony formation in tissue culture provides evidence for the presence of hematopoietic stem cells and early progenitor cells in the adult human liver. This would suggest that the adult human liver continues to contribute to hematopoiesis and may be an important site for the differentiation of lymphohematopoietic cells involved in disease states, such as autoimmune hepatitis and graft rejection after liver transplantation.     

The gp130-stimulating designer cytokine hyper-IL-6 promotes the expansion of human hematopoietic progenitor cells capable to differentiate into functional dendritic cells

OBJECTIVE: Hyper-IL-6, a fusion protein of interleukin-6 and its specific receptor, together with stem cell factor leads to the proliferation of primitive hematopoietic progenitor cells. Based on these findings, the current study examined whether hyper-IL-6 promotes the growth of precursor cells that can be further differentiated into dendritic cells in the presence of additional cytokines. METHODS: Dendritic cell cultures were generated from CD34(+) hematopoietic progenitor cells derived either from bone marrow or from peripheral blood. CD34(+) cells were cultured in the presence of cytokines for 2 weeks and then used for phenotyping and T-cell stimulation assays. RESULTS: Hyper-IL-6 in the presence of stem cell factor induced a 60- to 80-fold expansion of CD34(+) progenitor cells following 2 weeks of culture in serum-free medium. The addition of granulocyte-macrophage colony-stimulating factor to hyper-IL-6 and stem cell factor was essential for the differentiation of expanded progenitor cells into antigen presenting cells capable of inducing a primary T-cell response to soluble protein, which is a typical feature of dendritic cells. Phenotypic analyses confirmed the expansion of immature dendritic cells, which could be further differentiated into mature CD83(+) dendritic cells under the influence of interleukin-4, interleukin-1beta, tumor necrosis factor-alpha, and prostaglandin E(2). The capacity of expanded dendritic cells to stimulate protein-specific CD4(+) T cells was used to stimulate a primary T-helper cell response to the recombinant protein of the hepatitis-B core antigen in healthy donors. CONCLUSION: The expansion and differentiation of functional dendritic cells from CD34(+) progenitor cells under serum-free culture conditions allow for the possibility to develop more effective ways to immunize against viral infections and tumor diseases.     

Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity

Epithelial cells in embryonic day (ED) 12.5 murine fetal liver were separated from hematopoietic cell populations using fluorescence-activated cell sorting (FACS) and were characterized by immunocytochemistry using a broad set of antibodies specific for epithelial cells (alpha-fetoprotein [AFP], albumin [ALB], pancytokeratin [PanCK], Liv2, E-cadherin, Dlk), hematopoietic/endothelial cells (Ter119, CD45, CD31), and stem/progenitor cells (c-Kit, CD34, Sca-1). AFP(+)/ALB(+) cells represented approximately 2.5% of total cells and were positive for the epithelial-specific surface markers Liv2, E-cadherin, and Dlk, but were clearly separated and distinct from hematopoietic cells (Ter119(+)/CD45(+)). Fetal liver epithelial cells (AFP(+)/E-cadherin(+)) were Sca-1(+) but showed no expression of hematopoietic stem cell markers c-Kit and CD34. These cells were enriched by FACS sorting for E-cadherin to a purity of 95% as defined by co-expression of AFP and PanCK. Purified fetal liver epithelial cells formed clusters in cell culture and differentiated along the hepatocytic lineage in the presence of dexamethasone, expressing glucose-6-phosphatase (G6P) and tyrosine amino transferase. Wild-type ED12.5 murine fetal liver cells were transplanted into adult dipeptidyl peptidase IV knockout mice and differentiated into mature hepatocytes expressing ALB, G6P, and glycogen, indicating normal biochemical function. Transplanted cells became fully incorporated into the hepatic parenchymal cords and showed up to 80% liver repopulation at 2 to 6 months after cell transplantation. In conclusion, we isolated and highly purified a population of epithelial cells from the ED12.5 mouse fetal liver that are clearly separate from hematopoietic cells and differentiate into mature, functional hepatocytes in vivo with the capacity for efficient liver repopulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).     

生长因子及细胞外基质对肝前体细胞体外增殖及分化的影响

目的 明确多种生长因子对胚胎肝脏前体细胞体外增殖分化的影响。方法 用胶原酶从胎龄14．5d SD大鼠胚胎肝脏分离单个细胞，采用^3H-TdR掺入法检测生长因子对胎肝细胞体外增殖的促进作用，并观察生长因子及细胞外基质成分对胎肝干细胞集落形成的影响。采用免疫细胞双标记及G-6-P酶活性测定以检测肝前体细胞表面标记的表达及向成熟肝细胞的分化能力。结果 肝前体细胞在体外培养时显示克隆样生长的特性。促肝细胞生长因子(HGF)、表皮生长因子(EGF)能促进肝前体细胞的增殖，使DNA合成加速，并促进干细胞集落的形成及角蛋白19、白蛋白、G-6-P的表达。转化生长因子α对肝前体细胞的促增殖作用较弱。转化生长因子β对肝前体细胞的增殖起到抑制作用。细胞基质成分Ⅰ型胶原、Ⅳ型胶原、层黏连蛋白能促进肝干细胞集落的形成，而纤维连接蛋白的作用较弱。肝干细胞单克隆增殖需要生长因子及细胞外基质的共同参与，加入新鲜分离胎肝细胞培养液上清液时单细胞增殖较快，于第5天即形成细胞集落。结论 HGF、EGF对肝前体细胞的增殖分化起重要作用，细胞外基质成分亦参与了其增殖分化过程。肝干细胞单克隆培养除需生长因子和细胞外基质外，可能亦有某些造血细胞、间质细胞分泌的因子参与其增殖分化的调控。     

Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver

The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self-renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell-based analysis to examine precisely bilineage differentiation potential and self-renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133(+) cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self-renewing cell divisions. The clonogenic progeny of an LC-forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC-forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell-like capacity of isolated and clonally cultured CD133(+) LC-forming cells. Conclusion: Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis.     

Flow-cytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver

Stem cells responsible for tissue maintenance and repair are found in a number of organs. However, hepatic stem cells assumed to play a key role in liver development and regeneration remain to be well characterized. To address this issue, we set up a culture system in which primitive hepatic progenitor cells formed colonies. By combining this culture system with fluorescence-activated cell sorting (FACS), cells forming colonies containing distinct hepatocytes and cholangiocytes were identified in the fetal mouse liver. These cells express both CD49f and CD29 (alpha6 and beta1 integrin subunits), but do not mark for hematopoietic antigens such as CD45, TER119, and c-Kit. When transplanted into the spleen, these cells migrated to the recipient liver and differentiated into liver parenchymal cells. Our data demonstrate that hepatic progenitor cells are enriched by FACS and suggest approaches to supplanting organ allografting and improving artificial-organ hepatic support.     

Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells

Using a monoclonal antibody to murine CD38, we showed that a population of adult bone marrow cells that expressed the markers Sca-1 and c-kit but lacked the lineage markers Mac-1, GR-1, B220, IgM, CD3, CD4, CD8 and CD5 could be subdivided by the expression of CD38. We showed that CD38high c-kit+ Sca-1+, linlow/-cells sorted from adult bone marrow cultured with interleukin-3 (IL-3), IL-6, and kit-L produced much larger colonies in liquid culture at a greater frequency than their CD38low/- counterparts. In addition, we found that CD36low/ - cells contained most of the day-12 colony-forming units-spleen (CFU-S) but were not long-term reconstituting cells, whereas the population that expressed higher levels of CD38 contained few, but significant, day-12 CFU-S and virtually all the long-term reconstituting stem cells. Interestingly, the CD38high Sca-1+ c-kit+ linlow/- cells isolated from day-E14.5 fetal liver were also found to be long-term reconstituting stem cells. This is in striking contrast to human hematopoietic progenitors in which the most primitive hematopoietic cells from fetal tissues lack the expression of CD38. Furthermore, because antibodies to CD38 could functionally replace antibodies to Thy-1.1 in a stem cell purification procedure, the use of anti-CD38 may be more generally applicable to the purification of hematopoietic stem cells from mouse strains that do not express the Thy-1.1 allele.     

Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation

BACKGROUND & AIMS: Transformed hematopoietic stem/progenitor cells with an enhanced or acquired self-renewal capability function as leukemic stem cells. In a variety of solid cancers, stem/progenitor cells could be also targets of carcinogenesis. However, it remains unclear whether disruption of stem cell function directly contributes to cancer initiation. We sought to elucidate the mechanisms of self-renewal in hepatic stem/progenitor cells and the relation between stem cell function and hepatocarcinogenesis. METHODS: Functional analyses of polycomb-group protein Bmi1 and Wnt/beta-catenin, the molecules that are responsible for the self-renewal capability of many types of stem cells, were conducted in c-Kit(-)CD29(+)CD49f(+/low)CD45(-)Ter-119(-) hepatic stem/progenitor cells using retrovirus- or lentivirus-mediated gene transfer. The tumorigenicity of these cells transduced with the indicated retroviruses was also assessed by transplantation into nonobese diabetic/severe combined immunodeficient mice. RESULTS: Forced expression of Bmi1 and constitutively active beta-catenin mutant similarly promoted the self-renewal of hepatic stem/progenitor cells. The transplantation of Bmi1- or beta-catenin-transduced cells clonally expanded from single hepatic stem/progenitor cells produced tumors, which exhibited the histologic features of combined hepatocellular and cholangiocarcinoma. CONCLUSIONS: These observations imply that the dysregulated self-renewal of hepatic stem/progenitor cells serves as an early event in hepatocarcinogenesis, and they highlight the important roles of Bmi1 and the Wnt/beta-catenin pathway in regulating the self-renewal of normal or cancer stem cells in liver.     

Stem and progenitor cell systems in liver development and regeneration

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell‐specific surface markers including delta‐like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13⁺CD133⁺ fraction of human iPS‐derived cells contained numerous hepatic progenitor‐like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.     

Thrombopoietin stimulates murine lineage negative, Sca-1+, C-Kit+, CD34- cells: comparative study with stem cell factor or interleukin-3

It has recently been reported that human thrombopoietin (TPO) acts on early hematopoietic progenitor cells. Consequently, we investigated the effects of TPO on murine hematopoietic progenitor cells using lineage negative (Lin-), Sca-1+, c-Kit+ marrow cells from 5-fluorouracil-treated mice. One hundred enriched cells were cultured in suspension with various single cytokines for 5 days. When cultured with the single cytokines as stem cell factor (SCF), TPO, or interleukin (IL)-3, these cells were maintained or had increased by day 5, whereas only a few cells survived in cultures with granulocyte colony stimulating factor, IL-11, or IL-6. We extended the study in serum-free or serum-containing suspension cultures with SCF or TPO. Anti-TPO antibodies did not inhibit the effects of SCF on enriched cells but did inhibit the effects of TPO on those cells. We further examined the effects of TPO, SCF, and IL-3 on other populations of murine hematopoietic progenitor cells. Either TPO or SCF as a single cytokine could maintain murine Lin-, Sca-1+, c-Kit+, CD34- marrow cells, which are the most dormant cells. In addition, IL-3 increased Lin-, Sca-1-, c-Kit+ cells more than did SCF and TPO but did not stimulate Lin-, Sca-1+, c-Kit+, CD34- cells more. These results indicate that TPO as well as SCF may be key regulators in the proliferation of murine hematopoietic early progenitor cells.     

小鼠胚胎与成体肝脏中干细胞变化的探讨

目的 依据干细胞生物学特性,探讨从胚胎到成体肝脏内具干细胞特性细胞的变化状况.方法 分离不同胎龄与日龄小鼠肝脏细胞进行体外培养,观察分离获得的细胞数、培养形成的集落数及生长状态,并对集落进行干细胞表面标记物(c-kit、c-Met、Nestin)、肝细胞表面标记物(AFP、ALB)及胆管上皮细胞表面标记物(CK19)的标记.结果 各组分离的细胞经体外培养均有集落形成,肝干细胞的表面标记提示形成的集落具有肝干细胞特性.其中以第15天胚胎肝脏分离所获的细胞数和培养形成的集落数最多、生长状态最好.15 d后观察的各项指标渐减,细胞生长状态渐差.结论 早期胚胎肝脏中大部分是具有干细胞特性的细胞,随胎龄增加,有干细胞特性的细胞逐渐减少,直至出生第5天仅有少数保持未分化状态的细胞存留在肝脏内.     

Differential expression of lymphoid and myeloid markers on differentiating hematopoietic stem cells in normal and tumor-bearing adult human liver

The presence and phenotype of lineage-committed hematopoietic progenitors in the normal adult human liver (AHL) were investigated and compared with the profiles of differentiating hematopoietic precursor populations detected in liver bearing metastases of colonic origin. Levels of hematopoietic stem cells (HSCs) (CD34(+)CD45(+)) detected in hepatic mononuclear cell (HMNC) populations were increased 6-fold when compared with matched peripheral blood samples. In normal liver, less than 5% of HSCs expressed the myeloid-associated antigen, CD33, whereas considerable proportions expressed lymphoid-associated markers (T cell, 33.39%; B cell, 17.39%; and natural killer [NK] cell, 37.17%). Significant increases were observed in the relative proportions of hepatic HSCs coexpressing CD33 (20.53%; P =.001), and the T-cell marker (CD7, 58. 13%; P =.02) in tumor-bearing liver compared with normal liver. HSCs with B-cell progenitor phenotype (CD19(+)) were significantly decreased in tumor-bearing liver (0.06%; P =.02). Despite these differences, the activation status of hematopoiesis, as measured by the coexpression of the differentiation and activation markers, CD38 and CD45RA, did not differ significantly between normal and tumor-bearing liver. These results indicate that the normal AHL harbors lineage-committed hematopoietic progenitors, and the vast majority of these progenitors express lymphoid-associated antigens with changes occurring in both the myeloid and lymphoid compartments of the hepatic hematopoietic pathway on tumor challenge. While tumor-bearing livers are enriched for intrahepatic myeloid precursors and T-cell progenitor cells, further studies are required to establish the origin and in situ development potential of hepatic HSCs in the adult human and their role in tumor immunity.