A new culture medium for human skin epithelial cells

Summary A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.     

Variations in humanized and defined culture conditions supporting derivation of new human embryonic stem cell lines

The evolution of "humanized" (i.e., free of animal sourced reagents) and ultimately chemically defined culture systems for human embryo stem cell (hESC) isolation and culture is of importance to improving their efficacy and safety in research and therapeutic applications. This can be achieved by integration of a multitude of individual approaches to replace or eliminate specific animal sourced reagents into a single comprehensive protocol. In the present study our objective was to integrate strategies obviating reliance on some of the most poorly defined and path-critical factors associated with hESC derivation, namely the use of animal immune compliment to isolate embryo inner cell mass, and animal sourced serum products and feeder cells to sustain hESC growth and attachment. As a result we report the derivation of six new hESC lines isolated by outgrowth from whole blastocysts on an extracellular matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined serum-free medium (SFM) containing only human sourced and recombinant protein. Further, outgrowth of embryonic cells from whole blastocysts in both media could be achieved for up to 1 week without reliance on feeder cells. All variant conditions sustained undifferentiated cell status, a stable karyotype and the potential to form cells representative of all three germinal lineages in vitro and in vivo, when transitioned off of feeders onto Laminin or Matrigel. Our study thus demonstrates the capacity to integrate derivation strategies eliminating a requirement for animal immune compliment and serum products, with a transitional requirement for human feeder cells. This represents another sequential step in the generation of therapeutic grade stem cells with reduced risk of zoonotic pathogen transmission.     

Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage,blood outgrowth endothelial cells and cells from milk teeth

The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g. for children and minors) dental pulp cells from milk teeth represent a valuable alternative.     

Identification of human fibroblast cell lines as a feeder layer for human corneal epithelial regeneration

There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4) in a 35-mm dish (9.6 cm(2)) grew to confluence (about 1.87-2.41 × 10(6) cells) in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.     

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

We previously showed that Wnt3a could stimulate human embryonic stem （hES） cell proliferation and affect cell fate determination. In the absence of feeder cell--derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wntl and Wnt5a. While Wntl displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wntl enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wntl gene in immortalized human adult fibroblast （HAFi） cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt tmnsgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells （W3R） that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.     

人羊膜上皮细胞和胚胎干细胞

人胚胎干细胞有着巨大的医学应用前景,但人胚胎干细胞要求的生长条件很高,体外很难模拟其生长的体内环境,因此控制人胚胎干细胞的生长常不理想,而使用鼠胚胎成纤维细胞(MEF)作为滋养层则存在动物源性污染的问题。该文阐述人羊膜上皮细胞(HAEC)的特点及其作为滋养层培养胚胎干细胞的现状,并探讨基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用,为建立更优化的培养系统提供依据。     

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs,microRNAs and proteins among different feeders and conditioned media

Background  

Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages.     

以人皮肤成纤维细胞作为饲养层细胞培养人胚胎干细胞

目的：比较人皮肤成纤维细胞（humandermalfibroblasts,HDFs）与小鼠胚胎成纤维细胞（Mouseembryonicfibroblasts,MEFs）的增殖能力及研究人皮肤成纤维细胞作为饲养层支持人胚胎干细胞（humanembryonicstemcells,hESCs）未分化生长的能力。方法：利用组织贴壁法从人皮肤中分离出HDFs,通过细胞形态的观察和生长曲线的绘制比较HDFs与MEFs的体外增殖能力。将HDFs作为饲养层细胞与hESCs共培养,传代12代后,检测hESCs碱性磷酸酶（AKP）、表面特异性标志及胚胎干细胞特异性转录因子。结果：HDFs可连续传代培养15代以上,10代以下的HDFs增殖迅速,而MEFs自第4代起,增殖能力就明显下降;hESCs在HDFs饲养层上可传代培养12代以上,克隆边界清晰,细胞排列紧密,碱性磷酸酶、表面标志物检测均呈阳性,表达了hESCs特异性转录因子。结论：HDFs比MEFs具有更强的增殖能力;HDFs可作为培养hEscs的饲养层细胞。     

Effects of feeder layers made of human,mouse, hamster,and rat cells on the cloning efficiency of transformed human cells

Summary The effect of feeder layers on cloning efficiency of transformed human cells was investigated. Embryonic human skin or lung fibroblasts; adult human skin fibroblasts; early passage cells from embryos of mouse, rat, and hamster; established mouse cell lines; 3T3 and 10T1/2 were used as feeder layers after they were lethally exposed to Co-60 gamma-rays at 3,000 rad. As test cells to study the effect of feeder layers on cloning efficiency, WI-38 CT-1 cells transformed in vitro by Co-60 gamma-rays and HGC cells cultured from a human gastric cancer were used. The effect of feeder layers on the cloning efficiency of the test cells was dependent on cell density of feeder layer cells, sources of the feeder layer cells, and kinds of test cells. An optimal density of feeder cels produced cloning efficiencies 3 to 15 times higher than in cultures without a feeder layer. Generally, high density of cells in feeder layers decreased the cloning efficiency of the test cells, presumably owing to contact inhibition of growth and depletion of essential nutrients by the feeder layer cells. Regarding the effect of the feeder layers made of human fibroblasts, there were no significant differences in population doubling levels; tissue origins of fibroblasts; or fibroblasts derived from normal individuals, patients with cancer, or with a genetically high familial incidence of cancer, hereditary adenomatosis of the colon and rectum. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

Establishment of a long-term culture system for rat colon epithelial cells

Summary The aim of this study was to establish a long-term culture system for rat colon epithelial cells. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salts]-containing buffer, taken up in conditioned medium from the normal rat kidney fibroblast cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C-treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin 19 and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating factors on growth and gene expression of normal colon epithelial cells in vitro.     

Human feeder layers for human embryonic stem cells

Human embryonic stem (hES) cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast (MEF) feeder layers, which permit continuous growth in an undifferentiated stage. To use these unique cells in human therapy, an animal-free culture system must be used, which will prevent exposure to mouse retroviruses. Animal-free culture systems for hES cells enjoy three major advantages in the basic culture conditions: 1). the ability to grow these cells under serum-free conditions, 2). maintenance of the cells in an undifferentiated state on Matrigel matrix with 100% MEF-conditioned medium, and 3). the use of either human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder layers. In the present study, we describe an additional animal-free culture system for hES cells, based on a feeder layer derived from foreskin and a serum-free medium. In this culture condition, hES cells maintain all embryonic stem cell features (i.e., pluripotency, immortality, unlimited undifferentiated proliferation capability, and maintenance of normal karyotypes) after prolonged culture of 70 passages (>250 doublings). The major advantage of foreskin feeders is their ability to be continuously cultured for more than 42 passages, thus enabling proper analysis for foreign agents, genetic modification such as antibiotic resistance, and reduction of the enormous workload involved in the continuous preparation of new feeder lines.     

DNA Synthesis and Fos and Jun Protein Expression in Mitotic and Postmitotic WI-38 Fibroblasts in Vitro

Normal human embryonic lung fibroblasts WI-38 differentiate spontaneously along the cell lineage mitotic fibroblasts (MF) I, II, and III and postmitotic fibroblasts (PMF) IV, V, VI, and VII in the fibroblast stem cell system in vitro, when appropriate methods are applied. The mitotic fibroblasts can be induced to shift to postmitotic fibroblasts by two treatments with mitomycin C (2× MMC) in a short period of time compared to spontaneous development. Mitotic and postmitotic fibroblast cell types have specific morphological and biochemical properties, e.g., [³⁵S]methionine polypeptide markers in 2D PAGE. Spontaneously arisen and experimentally induced (2× MMC) PMF have the same morphological and biochemical characteristics. Mitotic fibroblasts have 2n DNA and undergo DNA synthesis for reduplication. Postmitotic cells undergo, on average, two rounds of DNA synthesis for endoreduplication (polyploidization). Spontaneously arisen and experimentally induced postmitotic populations are composed of postmitotic fibroblasts PMF IV, V, and VI with 2n, 4n, and 8n DNA. DNA synthesis of mitotic and postmitotic WI-38 cell populations may be regulated by the expression of Fos and Jun proteins. The Fos level of MFs was higher by a factor of 15-24 and the Jun level of MFs by a factor of 4.2-6.3 than those of spontaneously arisen PMFs. In 2× MMC-induced PMFs, the Fos level was about 4.4-7.5 times higher and the Jun level 1.7-3.3 times higher than that of spontaneously arisen PMFs. The down-regulation of these two parameters is a normal event in the development of mitotic to postmitotic WI-38 fibroblasts in the fibroblast stem cell system and is not related to cellular aging.     

High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na(+)-dependent taurine transporter TauT, and passive release via a volume-sensitive leak pathway. Here we demonstrate that: (i) TauT localizes to the primary cilium of growth-arrested NIH3T3 fibroblasts, (ii) long-term exposure to TNF(alpha) or hypertonic sucrose medium, i.e., growth medium supplemented with 100 mM sucrose, increases ciliary TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium.     

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 x 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5-13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells

Induced pluripotent stem(iPS) cells can be derived from human somatic cells by cellular reprogramming.This technology provides a potential source of non-controversial therapeutic cells for tissue repair,drug discovery,and opportunities for studying the molecular basis of human disease.Normally,mouse embryonic fibroblasts(MEFs) are used as feeder layers in the initial derivation of iPS lines.The purpose of this study was to determine whether SNL fibroblasts can be used to support the growth of human iPS cell...     

Protein factors which regulate cell motility

Summary Cell motility (i.e., movement) is an essential component of normal development, inflammation, tissue repair, angiogenesis, and tumor invasion. Various molecules can affect the motility and positioning of mammalian cells, including peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases. Recent studies have demonstrated a group of motility-stimulating proteins which do not appear to fit into any of the above categories. Examples include: 1)scatter factor (SF), a mesenchymal cell-derived protein which causes contiguous sheets of epithelium to separate into individual cells and stimulates the migration of epithelial as well as vascular endothelial cells; 2)autocrine motility factor (AMF), a tumor cell-derived protein which stimulates migration of the producer cells; and 3)migration-stimulating factor (MSF), a protein produced by fetal and cancer patient fibroblasts which stimulates penetration of three-dimensional collagen gels by non-producing adult fibroblasts. SF, AMF, and MSF are soluble and heat labile proteins with Mr of 77, 55, and 70 kd by SDS-PAGE, respectively, and may be members of a new class of cell-specific regulators of motility. Their physiologic functions have not been established, but available data suggest that they may be involved in fetal development and/or tissue repair.     

Effects of different extracellular matrices and co-cultures on human limbal stem cell expansion in vitro

To elucidate the effect of extracellular matrices (ECMs) and related and nonrelated-limbal feeder cells as substitutes for the in vivo niche on the phenotype and genotype of the limbal stem cell (SC) expansion in vitro, human limbal SCs were used. The limbus explants were expanded on human amniotic membrane (AM), commercial ECMs including matrigel (MAT), collagen (COL), and control (no ECM) in presence and absence of feeder cells including human limbal fibroblasts (LFs), a limbus-specific cell and mouse embryonic fibroblasts (MEFs). Proliferation, cell death, immunocytochemistry, expression of specific genes, ultrastructural characteristics, and size and granularity of expanded human limbal SCs in different groups were evaluated. The growth, cell proliferation, and survival of limbal SCs were enhanced by AM and MAT matrices. Ultrastructure and expression of stemness markers revealed that there was no significance difference between AM and MAT. However, flow cytometric analysis showed that the size and granularity of cultured cells increased in the presence of MAT and COL as well as in no ECM group. Moreover, co-culturing of limbal explants with LFs and MEFs on AM and MAT groups, enhanced the expansion and survival of cultured cells in comparison with others. In conclusion, the cultivation of human limbal explants on AM co-culturing with human LFs promises to be a good model for preparing undifferentiated epithelial sheets suitable for transplantation.     

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 × 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5–13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [³⁵S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.