Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro

During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.     

The in vitro and in vivo antitumor effects of pepleomycin alone or in combination with radiation

Pepleomycin is a derivative of bleomycin, which is less toxic to the host but possesses greater antitumor activity. Experiments are described here in which Chinese hamster V-79 cells in culture and a murine squamous cell carcinoma in vivo have been used to obtain survival curves for pepleomycin alone or in combination with radiation. In both systems the survival curves for pepleomycin alone are biphasic, and this drug proved to be twice as effective as bleomycin. Further, in contrast to bleomycin, which showed simple additivity with radiation, pepleomycin potentiated radiation injury to the tumor cells both in vivo and in vitro. Therefore, pepleomycin may be superior to bleomycin not only in drug therapy but also in combined modality therapy.     

Clonogenicity of mammalian cells in hybrid spheroids a new assay method

Summary A new in vitro method for determining the clonogenicity of mammalian cells in culture is described. The method is based on packaging clonogens into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates, termed hybrid spheroids, provide an in vivo-like environment for entrapped test cells, offering a realistic system for prospective tumor control studies. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of non-colony forming spheroids, the average number of clonogens per spheroid can be calculated, and the survival of irradiated test cells determined. In this fashion survival curves were obtained for HeLa, B-16 and HEp3 cells which corresponded to survival curves obtained in the conventional manner. The clonogenicity of cells, derived from a human maxillar melanoma surgical specimen was also determined by the hybrid spheroid method. With this method, plating efficiency increased in those cells which normally plate poorly, such as tumor cells, thus enabling survival measurements when this is not practical using conventional methods.     

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.     

Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro

By incubating multilayered primary cultures of human keratinocytes in low-calcium medium the suprabasal cell layers can be stripped off leaving a basal cell monolayer. When this monolayer is re-fed normal calcium medium a reproducible series of cell kinetic, morphological, and biochemical changes takes place resulting in the reestablishment of a multilayered tissue. Analysis of cell-cycle-specific proteins indicated that, during regeneration, a large cohort of cells became synchronized undergoing DNA replication after 3 days. Examination of culture morphology at the ultrastructural level confirmed the capacity of the basal cell monolayer to gradually reestablish a multilayered, differentiated epithelium. The ultrastructural appearance at 7 days poststripping was similar to that of unstripped cultures and was indicative of a tissue in steady state. Quantitation of cornified envelope formation at different times during regeneration showed that an increasing proportion of the cells were able to undergo terminal differentiation. In general, the pattern of keratin synthesis in the original epidermal explant labelled in vitro was similar to the pattern observed in human epidermis in vivo; however, in contrast to epidermis in vivo the explant also synthesized the hyperproliferative keratins 6 and 16. The in vitro differentiated keratinocytes showed underexpression of several proteins identified as differentiation markers, whereas several basal cell markers were overexpressed compared to the original explant. In addition, the in vitro differentiated keratinocytes synthesized some new proteins, notably keratins 7, 15 and 19. The basal layer remaining after stripping mainly expressed basal cell markers; however, during recovery, some of the differentiation-specific markers (e.g. keratin 10 and 15) were again expressed together with keratin no. 19, which is also expressed during wound healing in vivo. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model to investigate certain aspects of the regulation of epidermal tissue homeostasis.     

Wound closure in foetal rat skin.

Foetal rat skin rapidly closes an open wound in organ culture and in vivo, this possibly being unique to organs still in the morphogenetic stage. In the present study, examination was made of morphological changes in foetal rat skin during closure of open wounds inflicted at day 16 of gestation. Phase-contrast microscopy of open-wounded skin cultured in vitro indicated inward spreading of the peripheral skin to be responsible for wound closure. Wound closure in vitro was inhibited by cytochalasin B (10 micrograms/ml), not by hydroxyurea (2 mM), indicating prenatal wound closure to be mediated by regulation of the microfilament system rather than cell proliferation. During wound closure in vitro and in vivo, light and scanning electron microscopy of the peripheral skin showed cells in the periderm, the outermost layer of the foetal epidermis, to elongate centripetally and en masse, whereas the shape of underlying epidermal cells not to change. Numerous spindle-shaped cells and fibrous matrices in the mesenchyme were redistributed, becoming oriented along the wound edge. Following isolation of the mesenchyme and epidermis by treatment with Dispase and separate culturing, the capacity for wound closure in vitro was found to be retained only by the mesenchyme. Cellular activity within the mesenchyme, rather than in the epidermis, would thus appear essential to wound closure in foetal rat.     

Radiosensitivity of the cells of an established human melanoma cell line and the parent melanoma xenograft

Survival curves of cells from a human melanoma xenograft (E.F.) and a cell line (FME) established from this xenograft were determined. The cells of the established line were harvested from exponentially growing cultures, plateau phase cultures or solid tumours in athymic mice (FME-X) before irradiation. During irradiation the cells were kept suspended in culture medium. The colony forming ability of the cells was assayed in soft agar. The Do-value was significantly higher for the parent xenograft than for the established line, whether grown in vitro or in vivo (p less than 0.0001). In addition, the Dq-value was significantly lower for the xenograft than for exponentially growing cultures of the established line (p less than 0.05). Thus the radiation response of the cells of the established line was not representative for that of the cells from the parent xenograft. It is concluded that survival curves for established cell lines should be used with great caution in attempts to predict the radiocurability of human tumours of corresponding histological type.     

Generation of keratinocyte stem-like cells from human fibroblasts via a direct reprogramming approach

Skin repair and reconstruction are important after severe wound and trauma. Keratinocyte stem cells (KSCs) in the basal layer of the epidermis can regrow the stratified epidermis but are almost depleted after skin injury. Thus, generating enough KSCs is indispensable for skin regeneration. Pluripotent stem cells such as ESC and iPSC can differentiate into KSCs, but their applications are challenged by ethical issues and risks of tumor formation. Lineage reprogramming from one cell type into another one makes it feasible to generate the desired cell type. Here, we develop a method to convert human fibroblasts into induced keratinocyte stem-like cells (iKSC) by coupling transient expression of reprogramming factors with a chemically defined culture medium, without the formation of iPSC. iKSC resemble normal KSC in the morphological and phenotypic features and can differentiate in vitro and regenerate stratified epidermis after transplantation in vivo. Therefore, iKSC may provide abundant cellular sources for skin repair and regeneration.     

Differential chromosomal fluorescence with 33258 Hoechst

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10⁶ viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%.The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.     

Mouse epidermal cell cultures : II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin

In order to initiate studies on chemical carcinogenesis in an in vitro system analogous to mouse epidermis, primary epidermal cell cultures from perinatal mouse skin were established. A standardized method for the large-scale isolation of epidermal cells from late embryonic or newborn mouse dorsal skin has been developed. The epidermal cells were separated from fibroblasts by two series of discontinuous Ficoll density gradients. Using 80 to 100 animals/experiment, an average yield of 3×10⁶ viable epidermal cells/animal was obtained. The viability of the purified cell suspension exceeded 85%, and the plating efficiency, representing the growing cell fraction 24 h after plating, was about 43%.The cultures started as epithelial monolayers without fibroblast contamination. Their epidermal nature and origin was proved immunologically by an in vivo absorbed rabbit anti-mouse epidermis antiserum. The purity of the epidermal cells was quantitatively determined in trypsinized suspensions by the indirect immunofluorescence test yielding more than 95% epidermal antigen-positive cells. About half of the remaining antigen-negative cells could be identified as melanocytes. These highly purified epidermal cells grew in vitro for 2–3 weeks without dermal constituents or diffusible mesenchymal factors. The monolayers differentiated in culture giving rise to keratinizing cell sheets on top of the proliferating basal layer. By morphological, histochemical and physical methods, it could be evidenced that the differentiation processes in vitro are quite similar to keratinization in vivo.     

Human postnatal thymocytes generate phenotypically immature CD3dim, CD5dim, CD1abright progeny in organ culture.

We previously described an organ culture system that supports the in vitro development of human fetal thymocytes in murine alymphoid thymic rudiments without addition of exogenous factors. This approach to study human T cell precursors is limited by the requirement for fetal tissue. We show that human thymocytes from pediatric sources can be expanded under similar conditions. Organ cultures generated predominantly CD4 CD8 double positive cells, most of which maintained the phenotype CD3dim, CD5dim, and CD1abright, characteristic for double positive thymocytes ex vivo. This culture system should facilitate in vitro studies on human thymocyte development and repertoire selection.     

An in vitro rapid-turnover assay for human immunodeficiency virus type 1 replication selects for cell-to-cell spread of virus

We have developed a rapid-turnover culture system where the life span of a human immunodeficiency virus type 1-infected cell is controlled by periodic addition of a cytotoxic agent, mitomycin C. These mitomycin C-exposed cells are cocultured with a constant number of uninfected cells as new targets for the virus. Passage of the virus-infected cells under these conditions led to the emergence of a viral variant that was able to replicate efficiently in this culture system. After biologic and molecular cloning, we were able to identify a single frameshift mutation in the vpu open reading frame that was sufficient for growth of the mutant virus in the rapid-turnover assay. This virus variant spread more efficiently by cell-to-cell transfer than the parental virus did. Electron micrographs of cells infected with the delta vpu virus revealed a large number of mature viral capsids attached to the plasma membrane. The presence of these mature virus particles on the cell surface led to enhanced fusion and formation of giant syncytia with uninfected cells. Enhanced cell-to-cell transfer of the delta vpu virus provides an explanation for the survival of this mutant virus in the rapid-turnover culture system. The in vitro rapid-turnover culture system is a good representation of the in vivo turnover kinetics of infected cells and their continual replacement by host lymphopoietic mechanisms.     

Modeling the airway epithelium in allergic asthma: Interleukin-13-induced effects in differentiated murine tracheal epithelial cells

Summary Mucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.     

A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels

Malignant melanoma poses a serious health risk which is becoming more crucial as the incidence of this disease steadily increases. The development of appropriate in vitro models that reflect the in vivo tumor environment is a key factor for the study of this malignancy. The local tumor microenvironment plays a critical role in the ability of tumor cells to proliferate and metastasize. While interactions among various cell types are known to be important for tumor growth, most in vitro models utilize only tumor cells, ignoring the importance of tumor-stroma interactions, as well as the contribution of immune cells, which may be important for potential therapies. In addition, the cellular architecture found in vivo, known to be involved in changes in gene expression, is not reflected in standard two-dimensional culture systems. In this study, we have utilized rotating-vessel bioreactors to culture minced human melanoma specimens, allowing the culture of three-dimensional structures which reflect the cellular architecture and heterogeneous composition of the tumor site in vivo. The viability of the pieces in culture can be maintained for 1-2 wk. Immunohistochemical analysis shows multiple cellular types similar to the in vivo situation. Therefore, this system provides a unique model of human melanoma that mimics the in vivo tumor environment much more closely than current culture methods. This novel system may be utilized to determine the mechanism of action of current therapy protocols, as well as to develop new treatment regimens.     

Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation

We report here the isolation and characterization of three antisera, each of which is specific for a single keratin from one of the three different pairs (K1/K10, K14/K5, K16/K6) that are differentially expressed in normal human epidermis and in epidermal diseases of hyperproliferation. We have used these antisera in conjunction with monospecific cRNA probes for epidermal keratin mRNAs to investigate pathways of differentiation in human epidermis and epidermal diseases in vivo and in epidermal cells cultured from normal skin and from squamous cell carcinomas in vitro. Specifically, our results suggest that: (a) the basal-specific keratin mRNAs are down-regulated upon commitment to terminal differentiation, but their encoded proteins are stable, and can be detected throughout the spinous layers; (b) the hyperproliferation-associated keratin mRNAs are expressed at a low level throughout normal epidermis when their encoded proteins are not expressed, but are synthesized at high levels in the suprabasal layers of hyperproliferating epidermis, coincident with the induced expression of the hyperproliferation-associated keratins in these cells; and (c) concomitantly with the induction of the hyperproliferation-associated keratins in the suprabasal layers of the epidermis is the down-regulation of the expression of the terminal differentiation-specific keratins. These data have important implications for our understanding of normal epidermal differentiation and the deviations from this process in the course of epidermal diseases of hyperproliferation.     

Development of a bilayered living skin construct for clinical applications

An in vitro construct of human skin (living skin equivalent, LSE) has been engineered using serially passaged human epidermal keratinocytes and human dermal fibroblasts with a matrix of type I collagen. Cells are obtained from neonatal foreskin. LSE is cast, cultured, and shipped in a single culture insert. The size and shape of theinsert determines thesize and shape of the LSE. The dermal matrix consists of dermal fibroblasts within a condensed collagen lattice. The overlying epidermis is developed at the air-liquid interface to generate a protective cornified layer. Serum was not necessaryfor development of the epidermis. LSE for graft (Graftskin) has handling characteristics similar to split-thickness skin allowing it to be meshed, stapled, and sutured. LSE was cryopreserved using 65% glycerol an rapid freezing. Viability and in vivo performance on athymic mice were similar to fresh LSE. Cells derived from human eccrine gland were able to invade and form tubules rudimentary appendages may be possible.     

Dolichos biflorus agglutinin-binding site expression in basal keratinocytes is associated with cell differentiation

A basal layer of squamous epithelia such as epidermis contains stem cells, transit amplifying cells as well as postmitotic differentiating cells. A detailed knowledge of the transition among these cell types in the course of epidermal renewal is important. It would help in better understanding of many pathological processes, including cancer, and in employment of epidermal cells for therapeutic purposes. In this study we analyzed the possible role of Dolichos biflorus agglutinin (DBA)-reactive alpha-N-acetylgalactosamine glycosylation in behavior of the human epidermal basal cells under in vivo and in vitro conditions. The data received from porcine epidermis were also included. Part of basal cells was positive for DBA-binding sites and these cells exhibited a lower presence of beta1 integrin in their basal surface connected to the basement membrane. The perinuclear Golgi-like accumulation of beta1 integrin was observed in some cultured keratinocytes. The co-localization of integrin with DBA-binding sites and 58 kDa protein suggests that alpha-N-acetylgalactosamine glycosylation could be related to beta1 integrin retention in the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) at the beginning of the secretory pathway. The lack of anchorage in culture elevated the number of DBA-binding site positive cells without significant influence on cell growth when cells isolated directly from epidermis were employed in study. Some role of DBA-reactive glycoligand expressions in a suprabasal movement of differentiated basal cells can be hypothesized.     

Proliferation of human embryonic and fetal epidermal cells in organ culture

The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.     

Induction of Growth Factor Receptors on Cultured Human Melanocytes

Using a sensitive and selective culture system for human epidermal melanocytes, we have demonstrated that the morphologic changes induced by addition of phorbol 12-tetradecanoate 13-acetate (TPA) to proliferating newborn melanocytes are associated with induction of nerve growth factor (NGF) receptors, as measured by messenger RNA (mRNA) levels and protein accumulation and by cell surface immunofluorescent staining. Growth factor deprivation or addition of NGF similarly results in NGF receptor induction. NGF is believed to function in vivo and in vitro as a survival factor for many neural crest-derived cells and has been demonstrated to promote specific neural cell functions ranging from neurite outgrowth to enzyme induction, but to date no role for NGF has been identified with regard to normal human melanocytes. Our data demonstrate that, given appropriate stimulation, cultured human melanocytes may express the NGF receptor gene and therefore suggest that NGF may modulate human melanocyte behavior in vivo. This first demonstration of a growth factor receptor on human melanocytes provides an important opportunity to explore signal transduction relevant to their growth, differentiation, and malignant transformation.     

Epidermal receptor activator of NF-kappaB ligand controls Langerhans cells numbers and proliferation

Langerhans cells (LC) are the dendritic APC population of the epidermis, where they reside for long periods and are self-replicating. The molecular signals underlying these characteristics are unknown. The TNF superfamily member receptor activator of NF-kappaB ligand (RANKL, TNFSF11) has been shown to sustain viability of blood dendritic cells in addition to its role in promoting proliferation and differentiation of several cell types, notably osteoclasts. In this study, we have studied expression of the RANKL system in skin and have defined a key role for this molecule in LC homeostasis. In vitro and in vivo, human KC expressed RANKL and epidermal LC expressed cell surface RANK. In vitro, RANKL sustained CD34(+) progenitor-derived LC viability following 72-h cultures in cytokine-free medium (79.5 +/- 1% vs 55.2 +/- 5.7% live cells, respectively; n = 4; p < 0.05). In vivo, RANKL-deficient mice displayed a marked reduction in epidermal LC density (507.1 +/- 77.2 vs 873.6 +/- 41.6 LC per mm(2); n = 9; p < 0.05) and their proliferation was impaired without a detectable effect on apoptosis. These data indicate a key role for the RANKL system in the regulation of LC survival within the skin and suggest a regulatory role for KC in the maintenance of epidermal LC homeostasis.