Bipotent Differentiation Research Articles

In situ regeneration of bone-to-tendon structures: Comparisons between costal-cartilage derived stem cells and BMSCs in the rat model

Bone-tendon interface (BTI), also called enthesis, is composed of the bone, fibrocartilage, and tendon/ligament with gradual structural characteristics. The unique gradient structure is particularly important for mechanical stress transfer between bone and soft tissues. However, BTI injuries result in fibrous scar repairs and high incidences of re-rupture, which is attributed to the lack of local stem cells with tenogenic and osteogenic potentials. In the rat model, we identified unique stem cells from costal cartilage (CDSCs) with a high in situ regeneration potential of BTI structures. Compared to bone-marrow mesenchymal stem cells (BMSCs), CDSCs exhibit higher self-renewal capacities, better adaptability to low-oxygen and low-nutrient post-transplantation environments, as well as strong bi-potent differentiation abilities of osteogenesis and tenogenesis. After transplantation, CDSCs can survive, proliferate, and in situ gradually regenerate BTI structures. Therefore, CDSCs have a great potential for tissue engineering regeneration in BTI injuries, and have future clinical application prospects. Statement of significanceTissue engineering is a promising technique for bone-to-tendon interface (BTI) regeneration after injury, but it is still a long way from clinical application. One of the major reasons is the lack of suitable seed cells. This study found an ideal source of seed cells derived from costal cartilages (CDSCs). Compared to the traditional seed cell BMSCs, CDSCs have higher proliferation ability, strong chondrogenic and tenogenic differentiation potential, and better adaptability to low-oxygen and low nutrient conditions. CDSCs were able to survive, proliferate, and regenerate BTI structures in situ, in contrast to BMSCs. CDSCs transplantation showed strong BTI structures regeneration potential both histologically and biomechanically, making it a suitable seed cell for the tissue engineering regeneration of BTI.

The stemness of hepatocytes is maintained by high levels of lipopolysaccharide via YAP1 activation

BackgroundThe liver possesses a powerful regeneration ability, which is correlated with the stemness of hepatocytes in the portal vein (PV). However, the mechanism underlying the maintenance of hepatocyte stemness has not been elucidated. Here, we hypothesized that high levels of lipopolysaccharide from the portal vein might maintain the stemness of hepatocytes in the PV area.MethodsFirst, we examined the location of hepatic stem cells and the concentration of lipopolysaccharide (LPS) in the portal vein and inferior vena cava. Then, we assessed the effect of LPS on stemness maintenance in mice by using antibiotics to eliminate LPS and knocking out the LPS receptor, TLR4. In vitro, the effect of LPS on the stemness of hepatocytes was investigated by colony and sphere formation assays and assessment of pluripotent and stem cell marker expression. Furthermore, we studied the mechanism by which LPS regulates the stemness of hepatocytes. Finally, we ligated the portal vein branch to further verify the effect of LPS.ResultsWe found that a high level of LPS from the portal vein was correlated with the location of hepatic stem cells in the PV area, and elimination of LPS by antibiotics inhibited the expression of the stemness marker. LPS promoted colony and sphere formation and induced the upregulation of pluripotent and stem cell markers in AML12 cells. Furthermore, in the reprogramming medium, LPS facilitated the dedifferentiation of mature hepatocytes into hepatic progenitor-like cells, which exhibited a bipotent differentiation capacity in vivo and in vitro. Mechanistically, LPS bound TLR4 to regulate stemness of hepatocytes via the activation of YAP1 signaling, and blockade of YAP1 abolished the LPS-induced cell stemness and upregulation of pluripotent markers.ConclusionsOur study implies a correlation between LPS/TLR4/YAP1 signaling and cell stemness, and LPS was shown to be involved in stemness maintenance of hepatocytes in the PV area. LPS might be used to induce the dedifferentiation of mature hepatocytes into progenitor-like cells for repair of liver injury.

Generation of Hepatic Progenitor Cells from the Primary Hepatocytes of Nonhuman Primates Using Small Molecules.

Since primates have more biological similarities to humans than do other animals, they are a valuable resource in various field of research, including biomedicine, regenerative medicine, and drug discovery. However, there remain limitations to maintenance and expansion of primary hepatocytes derived from nonhuman primates. To overcome these limitations, we developed a novel culture system for primate cells. Primary hepatocytes from Macaca fascicularis (mf-PHs) were isolated from hepatectomized liver. To generate chemically derived hepatic progenitor cells (mf-CdHs), mf-PHs were cultured with reprogramming medium containing A83-01, CHIR99021, and hepatocyte growth factor (HGF). The bi-potent differentiation capacity of mf-CdHs into hepatocytes and biliary epithelial cells was confirmed by treatment with hepatic differentiation medium (HDM) and cholangiocytic differentiation medium (CDM), respectively. mf-PHs cultured with reprogramming medium showed rapid proliferation capacity in vitro and expressed progenitor-specific markers. Moreover, when cultured in HDM, these progenitor cells stably differentiated into hepatocyte-like cells expressing the mature hepatic markers. On the other hand, when cultured in CDM, the differentiated biliary epithelial cells expressed mature cholangiocyte characteristics. The results of the present study demonstrate that we successfully induced the formation of hepatic progenitor cells from mf-PHs by culturing them with a combination of small molecules, including growth factors. These results offer a means of expanding nonhuman primate hepatocytes without genetic manipulation for cellular resource, preclinical applications and regenerative medicine for the liver.

The Sca-1+ and Sca-1- mouse prostatic luminal cell lineages are independently sustained.

The phenotypic and functional heterogeneity of the mouse prostate epithelial cell lineages remains incompletely characterized. We show that the Sca-1+ luminal cells at the mouse proximal prostate express Sox2. These cells are replicative quiescent, castration resistant, and do not possess secretory function. We use the Probasin-CreERT2 and Sox2-CreERT2 models in concert with a fluorescent reporter line to label the Sca-1- and Sca-1+ luminal cells, respectively. By a lineage tracing approach, we show that the two luminal cell populations are independently sustained. Sox2 is dispensable for the maintenance of the Sca-1+ luminal cells but is essential for their facultative bipotent differentiation capacity. The Sca-1+ luminal cells share molecular features with the human TACSTD2+ luminal cells. This study corroborates the heterogeneity of the mouse prostate luminal cell lineage and shows that the adult mouse prostate luminal cell lineage is maintained by distinct cellular entities rather than a single progenitor population.

Prominin-1-expressing hepatic progenitor cells induce fibrogenesis in murine cholestatic liver injury.

Cholestatic liver injury is associated with intrahepatic biliary fibrosis, which can progress to cirrhosis. Resident hepatic progenitor cells (HPCs) expressing Prominin‐1 (Prom1 or CD133) become activated and participate in the expansion of cholangiocytes known as the ductular reaction. Previously, we demonstrated that in biliary atresia, Prom1(+) HPCs are present within developing fibrosis and that null mutation of Prom1 significantly abrogates fibrogenesis. Here, we hypothesized that these activated Prom1‐expressing HPCs promote fibrogenesis in cholestatic liver injury. Using Prom1CreERT2‐nLacZ/+;Rosa26Lsl‐GFP/+ mice, we traced the fate of Prom1‐expressing HPCs in the growth of the neonatal and adult livers and in biliary fibrosis induced by bile duct ligation (BDL). Prom1‐expressing cell lineage labeling with Green Fluorescent Protein (GFP) on postnatal day 1 exhibited an expanded population as well as bipotent differentiation potential toward both hepatocytes and cholangiocytes at postnatal day 35. However, in the adult liver, they lost hepatocyte differentiation potential. Upon cholestatic liver injury, adult Prom1‐expressing HPCs gave rise to both PROM1(+) and PROM1(‐) cholangiocytes contributing to ductular reaction without hepatocyte or myofibroblast differentiation. RNA‐sequencing analysis of GFP(+) Prom1‐expressing HPC lineage revealed a persistent cholangiocyte phenotype and evidence of Transforming Growth Factor‐β pathway activation. When Prom1‐expressing cells were ablated with induced Diphtheria toxin in Prom1CreERT‐nLacZ/+;Rosa26DTA/+ mice, we observed a decrease in ductular reactions and biliary fibrosis typically present in BDL as well as decreased expression of numerous fibrogenic gene markers. Our data indicate that Prom1‐expressing HPCs promote biliary fibrosis associated with activation of myofibroblasts in cholestatic liver injury.

476 Discovery and Characterisation of Bipotent, Angiogenic Haemangioblast Progenitor Cells in Adult Human Vasculature

The vascular adventitia contains progenitor cells and vasa vasorum, which play an important role in atherosclerosis. We previously discovered the presence of Lin-CD45+Sca-1+ colony forming unit-macrophage (CFU-M) progenitor cells in murine arterial adventitia. These progenitors have bipotent differentiation capacity for endothelial cells and macrophages, contributing to vasa vasorum expansion. Here we investigated for the existence of analogous progenitor cells in human vasculature. We first confirmed the presence of CFU-M progenitors in human vessels by culturing single cell digests from saphenous vein (SV) and left internal mammary artery (LIMA) in Methylcellulose for 14d. SV CFU-M yield was >50% greater than LIMA (p=0.0002). Fluoresce activated cell sorting of SV digests identified that CFU-M arise from a unique CD45+CD31-cKit+CD34+ subpopulation. Progenitor cells isolated from cultured CFU-M were plated in Matrigel and displayed angiogenic capacity by forming branching cords after 7d, containing endothelial cells and macrophages, indicating bipotent differentiation. As diabetes was associated with reduced recovery of CFU-M from human SV, we used streptozotocin-induced diabetic mice to study the effect of hyperglycaemia. Aortic CFU-M progenitors from diabetic mice displayed reduced clonogenic (p=0.006), self-renewal (p=0.028) and angiogenic capacity (p=0.048) compared to non-diabetic controls. Similar inhibitory effects were induced by exposing CFU-M progenitors from healthy mouse aorta to high glucose concentration in culture. Finally, hyperglycaemia and glucose conditioning also resulted in metabolic changes to CFU-M progenitors, including reduced mitochondrial respiration. Human vasculature contains CD45+CD31-cKit+CD34+ CFU-M progenitors which possess angiogenic capacity and haemangioblast-like, bipotent differentiation potential. Hyperglycaemia and increased glucose exposure inhibit the stem-like properties of these cells.

Functional Heterogeneity of Mouse Prostate Stromal Cells Revealed by Single-Cell RNA-Seq

SummaryWe perform a single-cell RNA sequencing analysis to investigate the phenotypic and functional heterogeneity of the adult mouse prostate stromal cells. Our analysis identifies three major cell populations representing the smooth muscle cells and two types of fibroblast cells enriched by Sca-1 and CD90. The Sca-1+CD90+ fibroblast cells are in direct contact with the epithelial cells and express growth factors and genes associated with cell motility, developmental process, and androgen biosynthesis. This suggests that they may regulate epithelial cell survival and growth. The Sca-1+CD90-/low myofibroblast-like cells highly express genes associated with the extracellular matrix and cytokine-mediated signaling pathways, indicating a role in tissue repair and immune responses. The Sca-1+CD90-/low cells significantly suppress the capacity of the basal cells for bipotent differentiation in the prostate organoid assay. Collectively, we identify the surface markers enabling physical separation of stromal subpopulations and generate the gene expression profiles implying their cellular functions.

Cells of Origin for Prostate Cancer.

Comprehensive knowledge of the normal prostate epithelial lineage hierarchy is a prerequisite to investigate the identity of the cells of origin for prostate cancer. The basal and luminal cells constitute most of the prostate epithelium and have been the major focuses of the study on the cells of origin for prostate cancer. Much progress has been made during the past few decades, mainly using mouse models, to understand the inter-lineage relationship and intra-lineage heterogeneity in adults as well as the lineage plasticity during conditions of stress. These studies have concluded that the adult mouse prostate basal and luminal cells are largely independently sustained under physiological conditions, but both types of cells possess the capacity for bipotent differentiation under stress or artificial experimental conditions. However, the existence or the identity of the putative progenitors within each lineage warrants further investigation. Whether the human prostate lineage hierarchy is completely the same as that of the mouse remains uncertain. Experiments from independent groups have demonstrated that both types of cells in mice and humans can serve as targets for transformation. But controversies remain whether the disease from distinct cells of origin display different clinical behaviors. Further investigation of the intra-lineage heterogeneity will provide new insights into this issue. Understanding the identity of the cells of origin for prostate cancer will help identify novel prognostic markers for early detection of aggressive prostate cancers, provide insights into the therapeutic vulnerability of these tumors, and inspire novel therapeutic strategies.

Enhanced Wnt/β-catenin and Notch signalling in the activated canine hepatic progenitor cell niche.

BackgroundThe liver has a large regenerative capacity. Hepatocytes can replicate and regenerate a diseased liver. However, as is the case in severe liver diseases, this replication may become insufficient or exhausted and hepatic progenitor cells (HPCs) can be activated in an attempt to restore liver function. Due to their bi-potent differentiation capacity, these HPCs have great potential for regenerative approaches yet over-activation does pose potential health risks. Therefore the mechanisms leading to activation must be elucidated prior to safe implementation in the veterinary clinic. Wnt/β-catenin and Notch signalling have been implicated in the activation of HPCs in mouse models and in humans. Here we assessed the involvement in canine HPC activation. Gene-expression profiles were derived from laser microdissected HPC niches from lobular dissecting hepatitis (LDH) and normal liver tissue, with a focus on Wnt/β-catenin and Notch signalling. Immunohistochemical and immunofluorescent studies were combined to assess the role of the pathways in HPCs during LDH.ResultsGene-expression confirmed higher expression of Wnt/β-catenin and Notch pathway components and target genes in activated HPC niches in diseased liver compared to quiescent HPC niches from normal liver. Immunofluorescence confirmed the activation of these pathways in the HPCs during disease. Immunohistochemistry showed proliferating HPCs during LDH, and double immunofluorescence showed downregulation of Wnt/β-catenin and Notch in differentiating HPCs. Vimentin, a mesenchymal marker, was expressed on a subset of undifferentiated HPCs.ConclusionsTogether these studies clearly revealed that both Wnt/β-catenin and Notch signalling pathways are enhanced in undifferentiated, proliferating and potentially migrating HPCs during severe progressive canine liver disease (LDH).

The stiffness and structure of three-dimensional printed hydrogels direct the differentiation of mesenchymal stromal cells toward adipogenic and osteogenic lineages.

The mechanical and physicochemical effects of three-dimensional (3D) printable hydrogels on cell behavior are paramount features to consider before manufacturing functional tissues. We hypothesize that besides good printability and cytocompatibility of a supporting hydrogel for the manufacture of individual tissues, it is equally essential to consider beforehand the desired tissue (bone, cartilage, fat). In light of its application, the structure and stiffness of printable hydrogel matrices influence cell geometry, which in turn impacts the differentiation fate. Embedded human mesenchymal stromal cells in printable type I collagen- and chitosan-agarose blends were induced to differentiate toward osteoblasts and adipocytes. Hydrogels' printability in air versus submerged printing in perfluorocarbon was evaluated according to the height, diameter, uniformity, and stability of 3D printed vertical cylinders. Bipotent differentiation within hydrogels was assessed histologically (morphology, cellularity), by immunohistochemistry (vimentin, smooth muscle actin), two-photon microscopy (spatial distribution), and real-time polymerase chain reaction (ALP, BGLAP, OPN, RUNX2, COL 1, aP2, PPARγ-2). Agarose and agarose blends revealed the most valid printability properties by generating uniform cylinders with an average height of 4 mm. Osteogenic differentiation was preferably achieved in anisotropic soft collagen-rich substrates, whereas adipogenic differentiation mostly occurred in isotropic stiff agarose-rich matrices. The conjugation of type I collagen to agarose with varying ratios is possibly a suitable bioink for a broad range of 3D printed mesenchymal tissues.

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

Self-Renewal and Differentiation Capacity of Urine-Derived Stem Cells after Urine Preservation for 24 Hours

Despite successful approaches to preserve organs, tissues, and isolated cells, the maintenance of stem cell viability and function in body fluids during storage for cell distribution and transportation remains unexplored. The aim of this study was to characterize urine-derived stem cells (USCs) after optimal preservation of urine specimens for up to 24 hours. A total of 415 urine specimens were collected from 12 healthy men (age range 20–54 years old). About 6×104 cells shed off from the urinary tract system in 24 hours. At least 100 USC clones were obtained from the stored urine specimens after 24 hours and maintained similar biological features to fresh USCs. The stored USCs had a “rice grain” shape in primary culture, and expressed mesenchymal stem cell surface markers, high telomerase activity, and normal karyotypes. Importantly, the preserved cells retained bipotent differentiation capacity. Differentiated USCs expressed myogenic specific proteins and contractile function when exposed to myogenic differentiation medium, and they expressed urothelial cell-specific markers and barrier function when exposed to urothelial differentiation medium. These data demonstrated that up to 75% of fresh USCs can be safely persevered in urine for 24 hours and that these cells stored in urine retain their original stem cell properties, indicating that preserved USCs could be available for potential use in cell-based therapy or clinical diagnosis.

Conversion of human fibroblasts to angioblast-like progenitor cells

Lineage conversion of one somatic cell type into another constitutes an attractive approach for research and clinical use. Lineage conversion can proceed in a direct manner, in the absence of proliferation and multipotent progenitor generation, or in an indirect manner, by the generation of expandable multipotent progenitor states. Here we report on the development of a combined reprogramming methodology that, transitioning through a plastic intermediate state, allows for the generation of human mesodermal progenitor cells while circumventing the traditional hallmarks of pluripotency. Converted mesodermal progenitor cells demonstrated bi-potent differentiation potential and were able to generate endothelial and smooth muscle lineages. Importantly, human fibroblasts can be converted into angioblast-like progenitor cells by non-integrative approaches. Differentiated angioblast-like cells exhibit neo-angiogenesis and anastomosis in vivo. The methodology for indirect lineage conversion to angioblast-like cells described here adds to the armamentarium of reprogramming approaches aimed at the clinical treatment of ischemic pathologies.

Implication of hepatic stem cells in functional liver repopulation

The liver has an enormous potential to restore the parenchymal tissue loss due to injury. This is accomplished by the proliferation of either the hepatocytes or liver progenitor cells in cases where massive damage prohibits hepatocytes from entering the proliferative response. Under debate is still whether hepatic stem cells are involved in liver tissue maintenance and regeneration or even whether they exist at all. The definition of an adult tissue-resident stem cell comprises basic functional stem cell criteria like the potential of self-renewal, multipotent, i.e. at least bipotent differentiation capacity and serial transplantability featuring the ability of functional tissue repopulation. The relationship between a progenitor and its progeny should exemplify the lineage commitment from the putative stem cell to the differentiated cell. This is mainly assessed by lineage tracing and immunohistochemical identification of markers specific to progenitors and their descendants. Flow cytometry approaches revealed that the liver stem cell population in animals is likely to be heterogeneous giving rise to progeny with different molecular signatures, depending on the stimulus to activate the putative stem cell compartment. The stem cell criteria are met by a variety of cells identified in the fetal and adult liver both under normal and injury conditions. It is the purpose of this review to verify hepatic stem cell candidates in the light of the stem cell definition criteria mentioned. Also from this point of view adult stem cells from non-hepatic tissues such as bone marrow, umbilical cord blood or adipose tissue, have the potential to differentiate into cells featuring functional hepatocyte characteristics. This has great impact because it opens the possibility of generating hepatocyte-like cells from adult stem cells in a sufficient amount and quality for their therapeutical application to treat end-stage liver diseases by stem cell-based hepatocytes in place of whole organ transplantation.

Human Hepatoma HepaRG Cell Line Engraftment in Severe Combined Immunodeficient × Beige Mice Using Mouse-Specific Anti-Fas Antibody

Chimeric mice with repopulated human hepatocytes are widely used for drug development research. HepaRG cell line is a naturally immortalized human liver cell line with progenitor properties and inducible bipotent differentiation capability. It would be useful if HepaRG cells could repopulate damaged livers severe, combined immunodeficient × beige (SCID/bg) mice and exhibit special human hepatic features. After inducing mouse hepatocyte apoptosis with an antimouse agonistic Fas monoclonal antibody (Jo2 mAb), HepaRG cells with bipotent differentiation capability were repopulated in SCID/bg mouse livers. HepaRG cells were transplanted intrasplenically into SCID/bg mice treated with 0.2 mg/kg Jo2 mAb once a week for 10 weeks. Human hepatocyte repopulation was characterized by immunohistochemistry for human serum albumin (HSA), Hep Par1, and CK18 and by immunofluorescence staining for HSA. Human albumin mRNA was detected by reverse-transcription polymerase chain reactions. HSA levels were quantified by Western blotting and enzyme-linked immunosorbent assays. Our results showed that HepaRG cell engraftment protected mice from the effects of Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. At 2 weeks after transplantation, increase concentrations of HSA were detected in recipient blood and liver. At 12 weeks after transplantation, approximately 15%–20% of mice showed livers repopulated with human hepatocytes. In conclusion, normal SCID/bg mice were suitable recipients for HepaRG cell transplantation when induced with Jo2 mAb. This chimeric mouse model with HepaRG cells could serve as a useful model for in vivo studies of drug metabolism and hepatitis virus infections.

Enhanced Self-Renewal Capability in Hepatic Stem/Progenitor Cells Drives Cancer Initiation

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. 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. 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. 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.

Plasticity of the hepatocyte phenotype in vitro: complex phenotypic transitions in proliferating hepatocyte cultures suggest bipotent differentiation capacity of mature hepatocytes

Plasticity of the hepatocyte phenotype in vitro: complex phenotypic transitions in proliferating hepatocyte cultures suggest bipotent differentiation capacity of mature hepatocytes

Plasticity of the hepatocyte phenotype in vitro: complex phenotypic transitions in proliferating hepatocyte cultures suggest bipotent differentiation capacity of mature hepatocytes.

Plasticity of the hepatocyte phenotype in vitro: complex phenotypic transitions in proliferating hepatocyte cultures suggest bipotent differentiation capacity of mature hepatocytes.

The axio-somatic model in embryonic and tumoral development

The growth and evolution of human tissues observed in embryogenesis and neoplasia are both reduced to a cellular theoretical axio-somatic model. The unlimited expansion and phenotype change of the model, are assured by the continuous generation of new cellular mitotic phenotypes which are grafted in a sequential order. Neogeneration results from the interaction between two cells of specific origins and situations: (a) axial cells of actual germ cell origin, generators and vectors of mitotic potential, (b) mitotically exhausted somatic cells of distal phenotypes, involved in meiotic recombinations, generating potential of neodifferentiation. The potentialities of both, axis and soma, are transformed by ‘fertilization’ in effectively growing new mitotic phenotypes. Bi-potent differentiation of germ line derived cells in gametopoiesis and haematopoiesis, is the source of the axial phenotype, identified in tissues as specific acid fast ‘lymphoid’ cells and/or vectors, formed by highly condensed hyperchromatinic nuclei and/or micronuclei endowed with DNAs of centromeric and kinetochoric types, wrapped in ceramide rich photo-affinity biomembranes.

Characterization of a Primitive Erythropoietic Progenitor Found in Mouse Marrow Before and After Several Weeks in Culture

The properties of a primitive mouse erythropoietic progenitor cell capable of macroscopic erythroid burst formation in methyl cellulose or agar have beert studied In assays of normal marrow. bursts that can achieve macroscopic size (approxi-mately 106 cells/burst) constitute a minor Proportion (30 %) of all bursts present after a 2-wk incubation. When normal marrow is first cultured in flasks containing serum but no added erythropoietin (Ep), within 2 wk macroscopic burst progenitors become the predominant erythropoietic cell type, and within 1 wk progenitors of isolated erythroblast Clusters (CFU-E) and small rapidly maturing bursts (day-3 BFU-E) are no longer detectable. The processes of formation and the compositions of macroscopic bursts from both normal and cultured marrow sources are identical. Macroscopic bursts have a late maturation pattern; in many, hemoglobinizing erythro-blasts are not found until 12-14 days after plating. Macroscopic burst formation is greatly enhanced when pokeweed-mito-gen-stimulated spleen-cell-conditioned medium (PWM-SCCM) is added to the assay culture, and this reduces the Ep requirement. However, even in the pre-sence of PWM-SCCM the Ep requirement is high; it equals or exceeds the concentra-tion of Ep required for late-maturing microscopic bursts. Morphologic analysis of 185 individual macroscopic bursts indicated that almost 90% contained megakaryo-cytes as well as erythroid progeny. In replating experiments, all types of erythropoietic precursors were detected in vary-ing numbers in 12-day-old macroscopic bursts. One of these actually gave rise to another two macroscopic bursts, and each of these contained megakaryocytes as well as erythroblasts. This confirms the extensive proliferative and bipotent differentiation capacities of macroscopic burst and suggests that they may also have self-renewal capabilities. Time course studies of the number of macroscopic burst progenitors present in the nonadherent fraction of marrow flask cultures showed these to be regularly and significantly increased after 2 wk. An association between the recovery of CFU-S and macroscopic burst progenitors was not ed. These results indicate that macroscopic burst formation may provide a simple end point for identifying a primitive uncommit-ted hemopoietic progenitor cell in normal adult marrow. The availability of a culture System that permits the production of this cell type, but restricts its subsequent differentiation, should be useful in future studies of early hemopoietic differentiation events.