Individual Keratinocytes Research Articles

Individual Keratinocyte Necroses in the Epidermis Are Apoptotic Keratinocytes in the Skin.

The patient was a 44-year-old woman with Stevens-Johnson syndrome due to receiving Baktar® (sulfamethoxazole trimethoprim) medication at our outpatient dermatology clinic. The epidermis, dermis, and subcutaneous adipose tissue samples showed numerous necrotic keratinocytes in the epidermis. Apoptotic nuclei were visualized as diaminobenzidine brown deposits with immunoperoxidase staining for cleaved caspase-3. The cleaved caspase-3-positive findings were consistent with eosinophilic material that appeared to be necrotic cells within the epidermis. Therefore, these eosinophilic materials may be apoptotic bodies. Generally speaking, eosinophilic cells are considered necrotic keratinocytes, especially in Japan. To the best of our knowledge, no studies have used apoptotic immunohistochemical markers to examine whether these structures are apoptotic in a human specimen.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Label-free quality control and identification of human keratinocyte stem cells by deep learning-based automated cell tracking.

Stem cell‐based products have clinical and industrial applications. Thus, there is a need to develop quality control methods to standardize stem cell manufacturing. Here, we report a deep learning‐based automated cell tracking (DeepACT) technology for noninvasive quality control and identification of cultured human stem cells. The combination of deep learning‐based cascading cell detection and Kalman filter algorithm‐based tracking successfully tracked the individual cells within the densely packed human epidermal keratinocyte colonies in the phase‐contrast images of the culture. DeepACT rapidly analyzed the motion of individual keratinocytes, which enabled the quantitative evaluation of keratinocyte dynamics in response to changes in culture conditions. Furthermore, DeepACT can distinguish keratinocyte stem cell colonies from non‐stem cell‐derived colonies by analyzing the spatial and velocity information of cells. This system can be widely applied to stem cell cultures used in regenerative medicine and provides a platform for developing reliable and noninvasive quality control technology.

Here comes the cavalry!

Cell Biology Healing small scratch wounds in the skin is much less well understood than the closing of deeper wounds. Bornes et al. used intravital microscopy to study the healing of scratches on the backs of mice. The authors followed individual keratinocytes as the cells multiplied and migrated to rapidly reestablish the skin's protective barrier. Basal keratinocytes multiplied at sites somewhat distant from the wound and then swarmed toward the wound, moving as a sheet of independently migrating cells. This allowed the replacement cells to bypass any intervening obstacles such as intact immobile hair follicles or sweat glands. The findings challenge a model proposing that the leading edge of migrating cells directly transmits forces to adjacent keratinocytes more distant from the wound bed to physically drag them along. Instead, leading-edge keratinocytes may initiate wound closure followed by individually moving keratinocytes within a cohesive layer. Life Sci. Alliance 4 , e202000765 (2020).

Scratch-induced partial skin wounds re-epithelialize by sheets of independently migrating keratinocytes.

Re-epithelialization is a crucial process to reestablish the protective barrier upon wounding of the skin. Although this process is well described for wounds where the complete epidermis and dermis is damaged, little is known about the re-epithelialization strategy in more frequently occurring smaller scratch wounds in which structures such as the hair follicles and sweat glands stay intact. To study this, we established a scratch wound model to follow individual keratinocytes in all epidermal layers in the back skin of mice by intravital microscopy. We discover that keratinocytes adopt a re-epithelialization strategy that enables them to bypass immobile obstacles such as hair follicles. Wound-induced cell loss is replenished by proliferation in a distinct zone away from the wound and this proliferation does not affect overall migration pattern. Whereas suprabasal keratinocytes are rather passive, basal keratinocytes move as a sheet of independently migrating cells into the wound, thereby constantly changing their direct neighboring cells enabling them to bypass intact obstacles. This re-epithelialization strategy results in a fast re-establishment of the protective skin barrier upon wounding.

Influence of magnetized abutment on biological seal of the implant–soft tissue interface by reconstructed human gingiva (RHG) model

Background: osseointegration in implants have received a lot of attention. Recently, the focus has extended towards the development of abutment materials supporting gingival soft tissue attachment. The attachment of the soft tissue to the implant/abutment surface is necessary to form a biological seal that protects the pathogenic microbial colonization which can lead to periimplantitis and bone resorption culminating in dental implant failure. Aim/Hypothesis: To evaluate the Influence of magnetized abutment surface on biological seal of the implant–soft tissue interface that would enable junctional epithelium(JE) attachment to the abutment through an internal basal lamina and hemidesmosomes in order to prevent the JE from migrating too in depth. Materials and Methods: We developed novel magnetic nanoparticles (MNs) coated with chitosan(CS) (CS-MNs) for enhancing cellular migration using magnetic force. CS-MNs were characterized by XRD, UV, SEM. The CS-MNs where spherical shape with a diameter from 10 nm to 15 nm. The magnetized abutments surface was treated with CS-MNs. Influence of CS-MNs treated abutment on reconstructed human gingiva (RHG) model consisting of differentiated gingival epithelium cells was assessed in terms of Epithelial attachment, down-growth along the abutment surface, quantitative analysis by permeability and cell attachment tests. Phenotype via histomorphology, scanning electron microscopy, and immunohistochemistry 10 days after implantation. Results: Abutment values obtained from the intraexperiment replicates including measurement of the left and right side of the abutment (total of four measurements) were first averaged, and Differences were determined using one-way ANOVA followed by Dunnett's uncorrected multiple comparisons test. Differences were considered significant at P < 0.05. histomorphometric analyses showed epithelial growth near perpendicular to the surface of abutments. The magnification clearly showed individual keratinocytes spread and attached to abutment surfaces via filopodia extensions. Conclusions and Clinical Implications: The current RHG model shows the active migration of epithelial cells near perpendicularly along the magnetized abutment surface with higher gingival fibroblast attachment as compared with an untreated surface. This novel system is thought to be useful in the development of biological seal around the implant and cell-based strategies for the repair or replacement of tissue and other novel therapies. Keywords: Soft tissue-implant interactions, biological seal, tissue-engineered oral mucosa, three-dimensional in vitro model

A 73-Year-Old With a Linear Rash

A 73-year-old man with a history of bilateral hip replacements developed a unilateral, asymptomatic skin eruption on his left buttock 3 months after a left hip revision. Over the ensuing 3 years, he noticed mild irritation with heat exposure. Hydrocortisone 1% cream provided no benefit. Skin examination revealed linear pink to brown papules with overlying scale, which did not involve his previous surgical scar site (Figure 1). The clinical differential diagnosis included linear lichen planus, lichen striatus, persistent postherpetic granulomatous skin eruption, or other linear skin entities. Punch biopsy showed acantholytic dyskeratosis. A diagnosis of segmental Darier disease (DD) was made (Figure 2).Figure 2Histopathology of the linear rash showed (A) focal acantholysis (black arrows) within the epidermis. B, Acantholysis is occurring just above the basal layer of the epidermis (yellow arrow). There is dyskeratosis with characteristic “corp ronds” (long black arrow) and “grains” (short black arrow). (Hematoxylin and eosin stain [A] 10x original magnification, [B] 20x original magnification.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) DD, a disease of keratinocyte adhesion, presents with grouped keratotic papules that are aggravated by heat and sweating and tends to develop during the third to fourth decades.1Burge S. Wilkinson J. Darier-White disease: a review of the clinical features in 163 patients.J Am Acad Dermatol. 1992; 27: 40-50Abstract Full Text PDF PubMed Google Scholar DD affects the body symmetrically. Segmental DD, however, presents with lesions arranged along the lines of Blaschko (segmental distribution). Diagnosis may be delayed because of the rarity of this entity. DD is an autosomal dominant genodermatosis caused by germline mutations in the ATP2A2 gene, which causes haploinsufficiency of epidermal calcium ATPase.2Sartori-Valinotti J. Peters M. Wieland C. Segmental type 1 Darier disease: a case series highlighting late-onset disease.Br J Dermatol. 2015; 173: 587-589Crossref PubMed Scopus (6) Google Scholar Segmental DD is thought to be caused by genetic mosaicism of the same gene. This mutation leads to keratinocyte discohesion and acantholytic dyskeratosis (rounding up and separation of individual keratinocytes, with accompanying abnormal keratinization), features that can be observed microscopically with skin biopsy (Figure 2).2Sartori-Valinotti J. Peters M. Wieland C. Segmental type 1 Darier disease: a case series highlighting late-onset disease.Br J Dermatol. 2015; 173: 587-589Crossref PubMed Scopus (6) Google Scholar, 3Harboe T. Willems P. Jespergaard C. Mølgaard M. Sørensen F. Bisgaard M. Mosaicism in segmental Darier disease: an in-depth molecular analysis quantifying proportions of mutated alleles in various tissues.Dermatology. 2011; 222: 292-296Crossref PubMed Scopus (15) Google Scholar, 4Pels I. Goodman M. Criteria for the histologic diagnosis of keratosis follicularis (Darier): report of a case with vesiculation.Arch Derm Syphilol. 1939; 39: 438-455Crossref Scopus (7) Google Scholar DD has a chronic course, and the goal of treatment is to limit exacerbations and prevent secondary infections.5Vogt K. Lohse C. El-Azhary R. et al.Kaposi varicelliform eruption in patients with Darier disease: a 20-year retrospective study.J Am Acad Dermatol. 2015; 72: 481-484Abstract Full Text Full Text PDF PubMed Google Scholar

524 Melanin localisation and transport in the human epidermis

Recent research has unraveled much about the control of melanosome movement in melanocytes (MC) and to a lesser extent how melanin granules are then transferred to neighboring keratinocytes (KC) in human epidermis. However, how melanin granules are distributed within KC is largely unknown despite this being critical for localised protection against UV damage. Here we examined how melanin is distributed within KC in human epidermis. Histocultures of human skin (photo-type II/III) were treated for 24h with nocodazole (microtubule polymerization inhibitor), monastrol (inhibitor of kinesin-5; important for spindle bipolarity) and cytochalasin B (actin filament polymerization inhibitor). Treatment effects were examined by expression of a) proliferation/mitosis (Ki67/pH3(Ser10)), b) differentiation (KRT10), c) centrosome localization (pericentrin), d) melanin movement (dynactin-1) and e) melanin localization (Warthin-Starry stain). The above treatments did not alter KC differentiation (KRT10). Nocodazole significantly increased mitosis detection in the surpabasal (but not basal) epidermis, and this concurred with distribution of total melanin predominating in the basal layer. Remarkably, the usual supranuclear distribution of melanin and centrosomes was reversed to a mostly subnuclear one, suggesting a previously-unreported association between the location of aggregated melanin and the centrosome. By contrast, monastrol increased mitosis detection in basal (but not suprabasal) epidermis. Now melanin was distributed suprabasally; still maintaining its supranuclear aggregation. This suggests that a) melanin can move between individual KC or b) MC can engage in more expansive melanin transfer beyond the basal layer. Cytochalasin-B did not alter mitosis detection but did disrupt aggregation of melanin granules, such that supranuclear melanin capping was now lost in the basal layer. Our results highlight a hitherto unappreciated role for centrosome, microtubule and actin function in the subcellular and intercellular melanin localization in human epidermis.

Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling.

Interfollicular epidermal (IFE) homeostasis is a major physiological process allowing maintenance of the skin barrier function. Despite progress in our understanding of stem cell populations in different hair follicle compartments, cellular mechanisms of IFE maintenance, in particular, whether a hierarchy of progenitors exists within this compartment, have remained controversial. We here used multicolour lineage tracing with Brainbow transgenic labels activated in the epidermis to track individual keratinocyte clones. Two modes of clonal progression could be observed in the adult murine dorsal skin. Clones attached to hair follicles showed rapid increase in size during the growth phase of the hair cycle. On the other hand, clones distant from hair follicles were slow cycling, but could be mobilized by a proliferative stimulus. Reinforced by mathematical modelling, these data support a model where progenitor cycling characteristics are differentially regulated in areas surrounding or away from growing hair follicles. Thus, while IFE progenitors follow a non-hierarchical mode of development, spatiotemporal control by their environment can change their potentialities, with far-reaching implications for epidermal homeostasis, wound repair and cancer development.

Drug-induced Skin Lesions in Cynomolgus Macaques Treated with Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulators

Three orally administered metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulators caused skin lesions consistent with delayed type-IV hypersensitivity in cynomolgus macaques in 2- and 12-week toxicity studies. Several monkeys developed macroscopic skin lesions in multiple locations after 8 to 9 days of dosing; the most prominent effects involved the genital region of males and generalized erythema occurred in both sexes. Microscopic lesions occurred in both clinically affected and unaffected areas and were characterized by lymphocytic interface inflammation, subepidermal bullae, and individual keratinocyte vacuolation/necrosis. In the 12-week study, clinical effects in 2 animals resolved with continued dosing, whereas in others the inflammatory process progressed with 1 female exhibiting systemic lymphocytic inflammation in multiple tissues. The inflammatory infiltrate consisted of CD3 and CD4 positive T lymphocytes with minimal CD68 positive macrophages and only rare CD8 positive T lymphocytes. A subset of animals given a dosing holiday was subsequently rechallenged with similar lesions developing but with a more rapid clinical onset. These skin lesions were consistent with type-IV delayed hypersensitivity with some features comparable to bullous drug eruptions in humans. A relationship between these findings and the intended mode of action for these compounds could not be ruled out, given the occurrence across different chemotypes.

Characteristics of keratinocytes in facial solar lentigo with flattened rete ridges: comparison with melasma.

Flattened rete ridges occurring on the face are not uncommon in solar lentigo (SL). To investigate the morphological changes of keratinocytes in facial SL with flattened rete ridges and melasma. In total, 25 patients with facial SL showing flattened rete ridges and 20 patients with melasma, in which rete ridge flattening is also a common feature, were included in the study. Skin biopsies were performed on lesional skin and perilesional normal skin. The histological findings showed that the epidermis was significantly thicker in SL skin with flattened rete ridges compared with perilesional normal skin. Comparative quantitative analysis of epidermal morphology revealed that the individual keratinocytes were larger in size in lesional skin, without a significant change in the number of cells. Anti-p16 antibody staining was more intense in lesional epidermis, suggesting senescence of keratinocytes in the thickened epidermis. By contrast, melasma samples showed no significant difference in epidermal thickness or keratinocyte morphology in terms of number or size compared with normal skin. These findings suggest that senescent changes in keratinocytes are important in the development of SL, even in the absence of rete ridge elongation, and the removal of keratinocytes harbouring melanin could be a possible strategy for SL treatment.

P2.005 Giant Condyloma Acuminata of Buschke and Lowenstein Treated Successfully Surgically

IntroductionThe Buschke Lowenstein tumour is an extremely rare, slow-growing, locally destructive, cauliflower-like mass, also known as giant condyloma acuminata.Case Report:We report a case of a 42 year old male who...

Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes

Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN-HIV vector showed the expected preference for transcribed genes while the SIN-MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN-MLV design has the lowest activity compared to both MLV and SIN-HIV vectors. This study indicates that SIN-MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders.

Vesiculobullous Skin Reaction Temporally Related to Firocoxib Treatment in a White Rhinoceros (Ceratotherium simum)

A 40 yr-old female white rhinoceros (Ceratotherium simum) suffered from chronic nail-bed abscesses. Due to worsening of clinical signs, the animal's nonsteroidal anti-inflammatory treatment was switched to firocoxib. Approximately 7 days after this change, the animal developed multifocal vesicles and bullae along the lateral aspects of the thorax and abdomen, the dorsum, and the proximal limbs. Cytology and culture did not identify an infectious etiology. Histologically, the lesions consisted of a severe, subacute vesiculobullous dermatitis with intraepidermal to subepidermal clefting with areas of individual keratinocyte necrosis and minor neutrophilic epidermal infiltrates. These findings are similar to those seen in some drug reactions in people; therefore an adverse drug reaction to the firocoxib was suspected.

Radiotherapy‐induced pemphigus vulgaris with autoantibodies targeting a 110 kDa epidermal antigen

Pemphigus is an autoimmune intraepidermal blistering disease mediated by autoantibodies targeting desmosomes. It can be induced by many triggers, such as ionizing radiation. We report a case of radiotherapy-induced pemphigus (RIP) with a review of the published cases in the English and French literature. A 61-year old man was diagnosed to have epidermoid carcinoma of the piriform sinus and then received a 70 Gy radiation therapy. One month after the treatment completion, multiple blisters and erosions occurred initially on the site of irradiation, then in other skin areas. Histological examination showed an intraepidermal blister with acantholysis and necrosis of individual keratinocytes. Direct immunofluorescence and indirect immunofluorescence were typical of pemphigus. Immunoblot revealed antibodies reacting with a 110 kDa antigen. This feature was consistent with the diagnosis of RIP. Less than 20 cases of RIP have been reported previously. Mean age at diagnosis was 64.2 years, and there is a slight female preponderance. RIP occurred, in most cases, initially within the area of irradiation. Our patient showed some distinctive findings never reported previously in RIP: a histological focal keratinocyte necrosis, and the presence of autoantibodies reacting with a 110 kDa keratinocytic protein in immunoblot analysis. Because of a different prognosis, it is important to differentiate RIP and paraneoplastic pemphigus (PNP), although cases of ionizing radiation-induced PNP had also been described. As in our patient, RIP seems to respond well to systemic corticosteroids and immunosuppressive therapy, which induce remission within a few months.

Visualization of plasmid delivery to keratinocytes in mouse and human epidermis

The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies.

In Vivo Imaging of Human and Mouse Skin with a Handheld Dual-Axis Confocal Fluorescence Microscope

Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.

The Calcium-Sensing Receptor-Dependent Regulation of Cell–Cell Adhesion and Keratinocyte Differentiation Requires Rho and Filamin A

Extracellular Ca(2+) (Ca(2+)(o)) functioning through the calcium-sensing receptor (CaR) induces E-cadherin-mediated cell-cell adhesion and cellular signals mediating cell differentiation in epidermal keratinocytes. Previous studies indicate that CaR regulates cell-cell adhesion through Fyn/Src tyrosine kinases. In this study, we investigate whether Rho GTPase is a part of the CaR-mediated signaling cascade regulating cell adhesion and differentiation. Suppressing endogenous Rho A expression by small interfering RNA (siRNA)-mediated gene silencing blocked the Ca(2+)(o)-induced association of Fyn with E-cadherin and suppressed the Ca(2+)(o)-induced tyrosine phosphorylation of β-, γ-, and p120-catenin and formation of intercellular adherens junctions. Rho A silencing also decreased the Ca(2+)(o)-stimulated expression of terminal differentiation markers. Elevating the Ca(2+)(o) level induced interactions among CaR, Rho A, E-cadherin, and the scaffolding protein filamin A at the cell membrane. Inactivation of CaR expression by adenoviral expression of a CaR antisense complementary DNA inhibited Ca(2+)(o)-induced activation of endogenous Rho. Ca(2+)(o) activation of Rho required a direct interaction between CaR and filamin A. Interference of CaR-filamin interaction inhibited Ca(2+)(o)-induced Rho activation and the formation of cell-cell junctions. These results indicate that Rho is a downstream mediator of CaR in the regulation of Ca(2+)(o)-induced E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.

Canine parvovirus-2b–associated erythema multiforme in a litter of English Setter dogs

Erythema multiforme (EM) was diagnosed in a litter of English Setter puppies. The puppies developed erythematous cutaneous lesions at the age of 2 weeks. Microscopically, there was individual keratinocyte apoptosis associated with lymphocyte exocytosis in all layers of the epidermis. Intranuclear viral inclusions were seen in multiple tissues and organs. Tissues from the tongue, lymph node, spleen, skin, and small intestine were positive for Canine parvovirus-2 (CPV-2) and negative for Canine distemper virus (CDV) and Canid herpesvirus 1 by fluorescent antibody test. Negative-staining electron microscopy detected parvovirus particles in the intestinal contents. The skin and small intestine were positive for CPV-2b and negative for CDV by polymerase chain reaction. The mucocutaneous junctions and small intestines stained positive for CPV by immunohistochemistry. The present report documents CPV-2b-associated EM in a litter of English Setters and substantiates the single previous report associating EM with CPV-2. The finding suggests that CPV should be considered as a possible cause of EM in dogs.

Growth dynamics of individual clones of normal human keratinocytes: observations and theoretical considerations

The life histories of 429 individual epidermal keratinocyte clones picked at random were studied. Individual basal keratinocytes were derived from asynchronous rapidly proliferating subconfluent cultures propagated in either a low calcium (0.1mM) or a high calcium (2mM) serum-free medium. Single-celled clones were isolated by seeding trypsin-EDTA dissociated cells into a Petri dish containing cloning chips. Chips with only one cell per chip were transferred into dishes containing either low calcium or high calcium growth factor replete serum-free medium. Clone formation was monitored microscopically and the number of cells in each colony tallied at least twice daily for further analysis. A total of 369 clones were established from seven different neonatal foreskin cell strains (A-F), and 60 clones were derived from one adult human skin cell strain (G). During a five-day culture interval, among 32 clones of strain A, 83% divided at least once, 50% divided once in 24 hours, 86% divided at least three times within three days, and more than 50% divided at least four to five times in five days. Of 231 clones amongst the other five cell strains (B-F), an average of 63% (±12 S,E) divided more than three times in an eight day period, the remainder divided either once, twice or not at all. Of the 106 clones of strain G, reared in high calcium serum-free medium, 67% divided more than three times in a six-day period, and 55% divided five or more times in 6 days. Clones derived from adult skin strain H had a lower clone forming potential with 70% dividing at least once in seven days, and only 30% dividing three or more times. By contrast, the average generation time (AvGT) for second and third passage keratinocytes derived from neonatal foreskin cultures was 24 hrs. Detailed dendrograms were constructed for many of the proliferating clones. The majority of clones expressed a synblastic division pattern with every cell dividing at least once per day. A fraction of clones either exceeded this circadian division rate or displayed a biphasic division pattern with all cells initially dividing once a day and then abruptly slowing to once every other day or to an intermediate rate. A minority of clones was committed to a few terminal divisions. The division patterns of the non-synblastic clones fit an alternating bifurcated branching mode of clonal expansion expressed by the Fibonacci sequence for numbers of accumulated cells per clone per day. These results were analyzed in terms of deterministic, probabilistic and a limit cycle oscillator models of cell division timing.