Primary cultures of human thymic epithelial tumors. Morphological and immunocytochemical characterization

Primary cultures are introduced as a method for an immunocytochemical and functional characterization of epithelial cells (ECs) from human thymic epithelial tumors. Neoplastic ECs were obtained after enzymatic digestion of the tumor tissue with dispase. The ECs were kept in culture for up to 1.5 months. Over this period a progressive decline in their proliferation rate was observed. In all five cases studied, ECs showed a co-expression of keratin and vimentin intermediate filaments in vitro as well as strong expression of major histocompatibility complex (MHC)-class I antigens and a progressive loss of MHC-class II antigens. Acetylcholine receptor (AchR)-epitopes were detected immunohistochemically by using monoclonal antibodies (mAbs) to the cytoplasmic site of the alpha-chain if the AchR. Epitopes were found in three of five thymomas in vivo and to a varying degree in all five cases in vitro.     

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34⁺ hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2–3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.     

Morphogenesis of mouse mammary epithelial cells growing within collagen gels: ultrastructural and immunocytochemical characterization

Mammary epithelial cells from adult virgin mice have been cultured within collagen gels in totally serum-free medium containing either epidermal growth factor or the mammogenic hormones, progesterone and prolactin, or prolactin alone. The cellular organization, differentiation and cell-type composition of the colonies from the three culture conditions were assessed by transmission electron microscopy and light-microscope immunocytochemistry. The epithelial cells form branching duct-like structures and, when exposed to mammogenic hormones, assume a secretory morphology (including casein micelles) similar to that seen in the early to mid-pregnant mouse.     

Cells derived from human umbilical cord blood support the long-term growth of undifferentiated human embryonic stem cells

Various types of human cells have been tested as feeder cells for the undifferentiated growth of human embryonic stem cells (hESCs) in vitro. We report here the successful culture of two hESC lines (H1 and H9) on human umbilical cord blood (UCB)-derived fibroblast-like cells. These cells permit the long-term continuous growth of undifferentiated and pluripotent hESCs. The cultured hESCs had normal karyotypes, expressed OCT-4, SSEA-4, TRA-1-60, and TRA-1-81, formed cystic embryonic body in vitro and teratomas in vivo after injected into immunodeficient mice. The wide availability of clinical-grade human UCB makes it a promising source of support cells for the growth of hESC for use in cell therapies.     

Primary cultures of hepatocytes on human fibroblasts

Summary Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Pattern of cytokine expression by rat liver epithelial cells supporting long-term culture of human CD34(+)umbilical cord blood cells

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis.     

Identification of subpopulations in mesenchymal stem cell-like cultures from human umbilical cord

Background

A variety of cell types can be identified in the adherent fraction of bone marrow mononuclear cells including more primitive and embryonic-like stem cells, mesenchymal stem cells (MSC), lineage-committed progenitors as well as mature cells such as osteoblasts and fibroblasts. Different methods are described for the isolation of single bone marrow stem cell subpopulations - beginning from ordinary size sieving, long term cultivation under specific conditions to FACS-based approaches. Besides bone marrow-derived subpopulations, also other tissues including human umbilical cord (UC) have been recently suggested to provide a potential source for MSC. Although of clinical importance, these UC-derived MSC populations remain to be characterized. It was thus the aim of the present study to identify possible subpopulations in cultures of MSC-like cells obtained from UC. We used counterflow centrifugal elutriation (CCE) as a novel strategy to successfully address this question.

Results

UC-derived primary cells were separated by CCE and revealed differentially-sized populations in the fractions. Thus, a subpopulation with an average diameter of about 11 μm and a small flat cell body was compared to a large sized subpopulation of about 19 μm average diameter. Flow cytometric analysis revealed the expression of certain MSC stem cell markers including CD44, CD73, CD90 and CD105, respectively, although these markers were expressed at higher levels in the small-sized population. Moreover, this small-sized subpopulation exhibited a higher proliferative capacity as compared to the total UC-derived primary cultures and the large-sized cells and demonstrated a reduced amount of aging cells.

Conclusion

Using the CCE technique, we were the first to demonstrate a subpopulation of small-sized UC-derived primary cells carrying MSC-like characteristics according to the presence of various mesenchymal stem cell markers. This is also supported by the high proliferative capacity of these MSC-like cells as compared to whole primary culture or other UC-derived subpopulations. The accumulation of a self-renewing MSC-like subpopulation by CCE with low expression levels of the aging marker senescence-associated β-galactosidase provides a valuable tool in the regenerative medicine and an alternative to bone-marrow-derived MSC.     

Mesenchymal stem cells from cryopreserved human umbilical cord blood

Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages, but the presence of mesenchymal stem cells (MSCs) in UCB has been disputed and it remains to be validated. In this study, we examined the ability of cryopreserved UCB harvests to produce cells with characteristics of MSCs. We were able to obtain homogeneous plastic adherent cells from the mononuclear cell fractions of cryopreserved UCB using our culture conditions. These adherent cell populations exhibited fibroblast-like morphology and typical mesenchymal-like immunophenotypes (CD73+, CD105+, and CD166+, etc.). These cells presented the self-renewal capacity and the mesenchymal cell-lineage potential to form bone, fat, and cartilage. Moreover, they expressed mRNAs of multi-lineage genes including SDF-1, NeuroD, and VEGF-R1, suggesting that the obtained cells had the multi-differentiation capacity as bone marrow-derived MSCs. These results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and therapeutic applications.     

Primary cultures of hepatocytes on human fibroblasts.

Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Characterization of mesenchymal stem cells isolated from the human umbilical cord

Numerous papers have reported that mesenchymal stem cells (MSCs) can be isolated from various sources such as bone marrow, adipose tissue and others. Nonetheless it is an open question whether MSCs isolated from different sources represent a single cell lineage or if cells residing in different organs are separate members of a family of MSCs. Subendothelial tissue of the umbilical cord vein has been shown to be a promising source of MSCs. The aim of this study was to isolate and characterize cells derived from the subendothelial layer of umbilical cord veins as regards their clonogenicity and differentiation potential. The results from these experiments show that cells isolated from the umbilical cord vein displayed fibroblast-like morphology and grew into colonies. Immunophenotyping by flow cytometry revealed that the isolated cells were negative for the hematopoietic line markers HLA-DR and CD34 but were positive for CD29, CD90 and CD73. The isolated cells were also positive for survivin, Bcl-2, vimentin and endoglin, as confirmed by RT-PCR and immunofluorescence. These cells can be induced to differentiate into osteogenic and adipogenic cells, but a new finding is that these cells can be induced to differentiate into endothelial cells expressing CD31, vWF and KDR-2, and also form vessel-like structures in Matrigel. The differentiated cells stopped expressing survivin, thus showing a diminished proliferative potential. It can be assumed that the subendothelial layer of the umbilical cord vein contains a population of cells with the overall characteristics of MSCs, with the additional capability to transform into endothelial cells.     

Differentiating characterization of human umbilical cord blood-derived mesenchymal stem cells in vitro

It has been demonstrated that the number and differentiating potential of bone marrow mesenchymal stem cells (MSCs) decrease with age. Therefore, the search for alternative sources of MSCs is of significant value. In the present study, MSCs were isolated from umbilical cord blood (UCB) by combining gradient density centrifugation with plastic adherence. Cultured cells were treated with ascorbate acid-2-phosphate, dexamethasone, beta-glycerophosphate dexamethasone, insulin, 1-methyl-3-isobutylxamthine, indomethacin, beta-mercaptoethanol, butylated hydroxyanisole, FGF-4 and HGF. Differentiating characterization of UCB-derived MSCs were detected by cytochemistry, immunocytochemistry, radioimmunoassay, RT-PCR and urea assay. The results showed UCB-derived MSCs could differentiate into osteoblasts, adipocytes and neuron-like cells. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on day 28 by morphology. Compared with the control, levels of AFP in the supernatant liquid increased significantly from day 12 and were higher on day 28 (P<0.01). Albumin increased significantly from day 16 (P<0.01). Urea was first detected on day 20 (P<0.01), and continued to increase on day 28 (P<0.01). Cells first expressed CK-18 on day 16 through immunocytochemistry analysis. RT-PCR analysis showed that differentiated cells could express a number of hepatocyte-specific genes in a time-dependent manner. Glycogen storage was first seen on day 24. Our results suggest that UCB-derived MSCs can differentiate not only into osteoblasts, adipocytes and neuron-like cells, but also into hepatocytes. Human UCB-derived MSCs are a new source of cell types for cell transplantation and therapy.     

Isolation, purification and characterization of hyaluronan from human umbilical cord residues

The main objective of this paper is to discuss new procedures of the isolation of Hyaluronan. Hyaluronic acid can be obtained from human umbilical cord residual, which is obtained from other biopharmaceutical productions. The route involves treatment of human umbilical cord residuals with sodium chloride solution, followed by ammonium quaternary salt solution precipitation; the solid is re-suspended in calcium chloride solution in order to dissociate the hyaluronan ammonium quaternary salt complex followed by ethanol-induced precipitation to give a product. The product was purified four times by chloroform extraction, and characterized by chemical methods such as the Blumenkrantz and Asboe-Hansen uronic technique for uronic acid determination, Elson Morgan qualitative tests for hexosamines, intrinsic viscosity, ion-exchange chromatography, and ¹³C NMR spectroscopy. The results showed that the product might be used in the formulation of ointment, lotion and cream for the treatment of skin diseases.     

Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney

Summary Epithelial cell cultures were prepared from normal human fetal kidney and established in long-term culture. The growth characteristics and production of keratin, and alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) activities were compared in a modified minimal essential medium (mMEM),d-valine-containing modified alpha-MEM (mALPHA) andl-valine mALPHA. The mean number of cumulative population doublings (CPDL) was significantly (P<0.001) enhanced with thel-valine mALPHA (40.8 CPDL) over that achievable in mMEM (14.2 CPDL) ord-valine mALPHA (18.3 CPDL) media. In all three media, greater than 95% of the cells in culture produced keratin throughout the life span of these cultures. Surface-associated fibronectin was absent in these cell cultures. AP and GGT activities increased as a function of subpassage and time in culture, with the greatest activity in thel-valine mALPHA. The expression of these renal cell-associated functions suggests that these cells in culture are proximal tubule epithelial cells. The conditions and procedures described in this paper can provide a human kidney epithelial cell culture system for studying human renal function, metabolism, cytotoxicity, genotoxicity, and transformation. Research was supported by a NIEHS (ES 3101) grant to S. M. D’Ambrosio and a NCI grant (CA21104) to J. E. Trosko.     

Stem cell-like properties of human umbilical cord lining epithelial cells and the potential for epidermal reconstitution

Background aimsStem cells are particularly attractive for many cell-based therapeutic interventions because of their ability to self-renew and their capacity to differentiate into site-specific differentiating cells. Restoration of the integrity of epithelial continuity is an essential aspect of wound repair and tissue regeneration. We are currently looking at the potential of human umbilical cord lining cells as a source of epithelial stem cells with appropriate differentiation capacity for potential epidermal reconstitution.MethodsWe isolated human umbilical cord lining epithelial cells (CLEC) and characterized their phenotype from the perspective of proliferative potential, telomere length, expression of epidermal differentiation markers, as well as stem cell-specific markers, and clonogenicity. Their potential for epidermal reconstitution was investigated in an organotypic culture model.ResultsThe results demonstrated that CLEC present a long telomere length and have a relatively high proliferative potential and passaging ability in culture. CLEC display some of the stem cell-specific markers for epithelial as well as pluripotent stem cells, including CK19, p63, OCT-4, SSEA-4, TRA-1–60, SOX2 and Nanog. CLEC are capable of generating a fully stratified epithelium in organotypic culture.ConclusionsThe potential of CLEC to be used in clinical applications for specialized epithelial reconstruction is still unexplored. The demonstration that CLEC have stem cell-like properties and are capable of generating fully stratified epithelium provides support for their potential clinical application in epidermal reconstitution.