In vitro screening system for hepatotoxicity: Comparison of bone‐marrow‐derived mesenchymal stem cells and Placenta‐derived stem cells

Stem cells have unique properties such as self‐renewal, plasticity to generate various cell types, and availability of cells of human origin. The characteristics are attentive in the toxicity screening against chemical toxicants. Placenta‐derived stem cells (PDSCs) have been spotlighted as a new cell source in stem cell research recently because they are characterized by their capacity to differentiate into multilineages. However, the use of PDSCs as an in vitro screening model for potential drug candidates has not yet been studied. Here, we analyzed the potentials for bone‐marrow‐derived mesenchymal stem (BM‐MSCs), which is a representative adult stem cells and PDSCs as an in vitro hepatotoxicity screening system, using well‐known hepatotoxicants. BM‐MSCs and PDSCs were analyzed to the potential for hepatogenic differentiation and were cultured with different concentrations of hepatotoxicants for time courses. The viability and ATP‐binding cassette (ABC) transporters were measured by the MTT assay and RT‐PCR, respectively. The sensitivities of PDSCs to hepatotoxicants are more sensitive than those of BM‐MSCs. The viability (IC₅₀) to in PDSCs was less than that of BM‐MSCs after 48 and 72 h (P < 0.05) of CCl₄ exposure. The toxicities of CCl₄ were decreased by fourfold in hepatogenic differentiation inducing PDSCs compared to the undifferentiated cells. The alteration of ABCGs was observed in PDSCs during differentiation. These findings suggest that the naïve PDSCs expressing ABCGs can be used as a source for in vitro screening system as well as the expression patterns of ABCG1 and ABCG2 might be involved in the sensitivity of PDSCs to hepatotoxicants. J. Cell. Biochem. 112: 49–58, 2011. © 2010 Wiley‐Liss, Inc.     

Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum

Objectives: Translational research using adult stem cells derived from various tissues has been highlighted in cell‐based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta‐derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. Materials and methods: Placental extract, extracted using water‐soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up‐take, PAS (Periodic Acid‐Schiff) staining and urea production were also analysed. Results: The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte‐specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. Conclusions: Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human‐based medium, in translational research for regenerative medicine.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Single‐cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live‐cell intracellular calcium flux was visualized through Fluo‐4‐AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37–2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45–3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub‐classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.     

Characterization of smooth muscle differentiation of purified human skeletal muscle‐derived cells

The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle‐derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low‐serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT‐PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α‐smooth muscle actin (ASMA). The cells cultured in low‐serum medium for 4 weeks also expressed ASMA, while the control group did not. RT‐PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.     

Telomerase repeat amplification protocol (TRAP): A new molecular marker for parathyroid carcinoma.

Telomerase results to be active in human germ, stem cells, several malignant cell tumors and in immortalized cell lines. In order to investigate if molecular mechanisms other than Rb gene inactivation can be helpful to diagnose malignancy of parathyroid tumors, we decided to investigate the presence of active telomerase in homogenates from different pathological parathyroid tissues (hyperplastic, adenomatous, carcinomatous, and normal) and primary cell cultures. The TRAP assay was performed to detect this activity in histologically characterized normal, hyperplastic, adenomatous, and carcinomatous human parathyroid tissues, primary cell lines, and one metastatic tissue from parathyroid carcinoma. Only malignant parathyroid glands and the metastatic tissue were TRAP positive. Our findings suggest that telomerase expression could represent an important molecular mechanism underlying the acquisition and progression of an aggressive phenotype of epithelial parathyroid cells and it may help to predict their malignant potential. The TRAP assay is easy to perform and it could become an additional tool to be included in the harmamentarium for the molecular diagnosis of parathyroid carcinoma.     

Comparison of Surface Markers between Human and Rabbit Mesenchymal Stem Cells

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.     

Human chorionic-plate-derived mesenchymal stem cells and Wharton’s jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells

Placenta-derived stem cells (PDSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal and differentiation and their immunomodulatory properties. Although many studies have characterized various PDSCs biologically, the properties of the self-renewal and differentiation potential among PDSCs have not yet been directly compared. We consider the characterization of chorionic-plate-derived mesenchymal stem cells (CP-MSCs) and Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) among various PDSCs and the assessment of their differentiation potential to be important for future studies into the applicability and effectiveness of PDSCs in cell therapy. In the present study, the capacities for self-renewal and multipotent differentiation of CP-MSCs and WJ-MSC isolated from normal term placentas were compared. CP-MSCs and WJ-MSCs expressed mRNAs for the pluripotent stem cell markers Oct-4, Nanog, and Sox-2. Additionally, HLA-G for immunomodulatory effects was found to be expressed at both the mRNA and protein levels in both cell types. The CP-MSCs and WJ-MSCs also had the capacities to differentiate into cells of mesodermal (adipogenic and osteogenic) and endodermal (hepatogenic) lineages. Expression of adipogenesis-related genes was higher in CP-MSCs than in WJ-MSCs, whereas WJ-MSCs accumulated more mineralized matrix than CP-MSCs. The WJ-MSCs expressed more of CYP3A4 mRNA, a marker for mature hepatocytes, than CP-MSCs. Thus, we propose that CP-MSCs and WJ-MSCs are useful sources of cells for appropriate clinical applications in the treatment of various degenerative diseases.     

Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.     

Orderly and nonstochastic acquisition of CD94/NKG2 receptors by developing NK cells derived from embryonic stem cells in vitro

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.     

T cell receptor-independent CD4 signalling: CD4-MHC class II interactions regulate intracellular calcium and cyclic AMP

CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II beta2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP.     

Abnormal calcium distribution in human parathyroid adenomas as possible cause of primary hyperparathyroidism

Current knowledge suggests that normal parathyroid glands and parathyroid adenomas have different sensitivities to environmental calcium. In search for morphological equivalents, 5 normal human and 10 porcine parathyroid glands, as well as 10 human parathyroid adenomas were investigated with regard to intracellular and extracellular calcium distribution. The glands were incubated for 2, 4, 6 and 20 h in tissue cultures using HAM's F10 medium with various calcium concentrations. For visualization of the calcium distribution in the tissue the method of pyroantimonate precipitation was applied. Specificity of the reaction was controlled by X-ray microanalysis. Shifts of the calcium pyroantimonate precipitates were quantitated by morphometry using an area-counting system. The results demonstrate that in normal parathyroid glands calcium precipitates are distributed randomly. Incubation of normal glands in medium with low calcium concentration (0.6 mM) provoked reduced amounts of intracellular and extracellular calcium complexes. When the incubations were performed in medium with high calcium content (2.6 mM), calcium accumulated inside parathyroid and stroma cells. In contrast to normal parathyroid glands, parathyroid adenomas fixed immediately after surgery showed an atypical calcium distribution with low amounts of intracellular and high amounts of extracellular calcium grains. The data suggest that in normal parathyroid glands the intracellular calcium concentration follows the extracellular environmental calcium concentration. Thus, calcium modulates parathyroid hormone (PTH) secretion via intracellular regulatory mechanisms. In parathyroid adenomas the calcium transport via the tumor cell membrane appears to be disturbed resulting in lowered intracellular calcium levels. This is remarkable since the environmental calcium concentration is elevated due to the hypercalcemia of primary hyperparathyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

甲状旁腺高血压因子和甲状旁腺素对细胞钙通道的不同作用

1989年Lewanczuk等[1]报道在自发性高血压大鼠（spontaneouslyhvnertensiverat,SHR）的血浆中发现一种具有独特升压效应的高血压因子．通过激活平滑肌细胞膜钙通道,提高细胞内游离钙（[Ca2 ]）水平起作用．随之证明这种循环血中的高血压因子来源于甲状旁腺,故称“甲状旁腺高血压因子（Parathyroidhypertensivefactor,PHF）”[2]。我们经实验研究证明,当给SD（Sprague-Dawley）大鼠注射经透析的SHR血浆后30min其血压开始升高,45min达高峰,60min恢复到注射前水平．同时经透析的血浆可使SD大鼠尾动脉条的细胞45Ca2 摄取增加,其…     

CD34 Class III positive cells are present in atherosclerotic plaques of the rabbit model of atherosclerosis

CD34 is a positive marker for haematopoietic stem cells and endothelial cells. Recent evidence suggests that haematopoietic progenitor cells are involved in atherogenesis. CD34-positive haematopoietic progenitor cells have never been described in rabbit atherosclerotic tissues. The aim of this study is to identify CD34-positive haematopoietic progenitor cells in rabbit atherosclerotic tissues, and to compare this with macrophage (RAM-11), alpha smooth muscle cell actin and fibroblast (prolyl-4-hydroxylase) immunoreactive cells. Sixteen Male New Zealand White rabbits were divided into two groups: Group 1, control diet (Con); group 2, 0.5% cholesterol diet, and killed after 12 weeks. Immunohistochemistry was used to detect CD34 haematopoietic progenitor cells. CD34-positive haematopoietic progenitor cells were identified both within and overlying atherosclerotic plaques. As well, these haematopoietic progenitor cells also stained for RAM-11, CD45, prolyl-4 hydroxylase and alpha smooth muscle cell actin. These findings suggest that in the rabbit model of atherosclerosis, the previously identified macrophages, smooth muscle cells and fibroblasts within and overlying atherosclerotic plaques might be of haematopoietic origin.     

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.