Clonal analysis of human clonogenic keratinocytes

Enzo, Elena; Cattaneo, Camilla; Consiglio, Federica; Polito, Maria Pia; Bondanza, Sergio; Luca, Michele De

doi:10.1016/bs.mcb.2022.02.009

Cited by 11 publications

(5 citation statements)

References 31 publications

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“…A clinically validated method specifically used for the in vitro culture of human primary keratinocytes consists of a biopsy cleaning step to remove the adipose tissue and partially the dermis. Then, the tissue is minced and incubated in a trypsinizing flask with a trypsin/EDTA solution (mixture of trypsin, chymotrypsin, and elastase) for 30 min at 37 • C. Cells are recovered after each round of trypsinization and plated onto a lethally irradiated feeder layer [23]. This procedure allows the collection of both interfollicular and follicular keratinocytes, including SCs used for cell and gene therapy applications (unpublished data and [10,24,25]).…”

Section: Skin Biopsy Processingmentioning

confidence: 99%

“…To gain insight into clone-founding cells, scRNA-seq was applied to the clinical-grade culture of human primary keratinocytes extracted from two healthy donors' truncal skin biopsies (Table 1, Figure 1C) [23] The transcriptomic profiles of 7354 cells were analyzed. Three clusters expressed high levels of clonogenic/basal markers (KRT14 + , TP63 + , ITGA6 + , and ITGB1 + ), whereas differentiated cells, identified as clusters TD1 and TD2, expressed SERPINB3, SFN, KRT10, IVL, and SPINK5.…”

Section: Single-cell Molecular Profiling Of In Vitro Cultured Human P...mentioning

confidence: 99%

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Decoding the Human Epidermal Complexity at Single-Cell Resolution

Polito

Marini

Palamenghi

et al. 2023

IJMS

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The epidermis is one of the largest tissues in the human body, serving as a protective barrier. The basal layer of the epidermis, which consists of epithelial stem cells and transient amplifying progenitors, represents its proliferative compartment. As keratinocytes migrate from the basal layer to the skin surface, they exit the cell cycle and initiate terminal differentiation, ultimately generating the suprabasal epidermal layers. A deeper understanding of the molecular mechanisms and pathways driving keratinocytes’ organization and regeneration is essential for successful therapeutic approaches. Single-cell techniques are valuable tools for studying molecular heterogeneity. The high-resolution characterization obtained with these technologies has identified disease-specific drivers and new therapeutic targets, further promoting the advancement of personalized therapies. This review summarizes the latest findings on the transcriptomic and epigenetic profiling of human epidermal cells, analyzed from human biopsy or after in vitro cultivation, focusing on physiological, wound healing, and inflammatory skin conditions.

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Section: Skin Biopsy Processingmentioning

confidence: 99%

Section: Single-cell Molecular Profiling Of In Vitro Cultured Human P...mentioning

confidence: 99%

Decoding the Human Epidermal Complexity at Single-Cell Resolution

Polito

Marini

Palamenghi

et al. 2023

IJMS

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“…The downregulation of PITX2 was also demonstrated by comparing oral mucosa holoclones to those derived from other epithelia, such as the limbus, conjunctiva, epidermis and urethra (Figure 2D). We further analyzed PITX2 expression in oral mucosa meroclones, which are the progeny of the transient amplifying cells (TA-cells) and retain a lower clonogenicity if compared to holoclones [23,24]. The results showed that PITX2 mRNA did not correlate with the clonogenicity of the clones (Figure 2E).…”

Section: Pitx2 Mrna Is Overexpressed In Oral Mucosa Compared To Ocula...mentioning

confidence: 99%

“…In this assay, a small aliquot of cells was cultured for 12 days onto a 100 mm dish, then fixed and stained with rhodamine B for the classification of clonal type. This was determined by the percentage of aborted colonies formed by the progeny of the founding cell [23,24]. When 0-5% of colonies were abortive, the clone was scored as holoclone (stem cell).…”

Section: Clonal Analysis and Colony-forming Efficiency Assaymentioning

confidence: 99%

Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up

Attico,

Galaverni,

Torello

et al. 2023

IJMS

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Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas.

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“…Although a human holoclone molecular signature is thus emerging, the most trustworthy assay able to define the clonal composition of an epidermal culture is still the clonal analysis (Enzo et al 2022). Further development of single-cell genetic and epigenetic analyses should give more insights that could allow to prospectively distinguish epidermal holoclones from the other clonal types.…”

Section: Advanced Therapies For Epidermolysis Bullosamentioning

confidence: 99%

Stairways to Advanced Therapies for Epidermolysis Bullosa

Rosa

Enzo

Palamenghi

et al. 2022

Cold Spring Harb Perspect Biol

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Epidermolysis bullosa (EB) is a devastating genetic skin disease typified by a plethora of different phenotypes and ranking from severe, early lethal, to mild localized forms. Although there is no cure for EB, recent progress in pharmacology and molecular and cellular biology is boosting the development of new advanced therapeutic strategies. Here we will focus on two main categories of such therapies: (1) those aimed at controlling inflammation and inducing reepithelialization of the wounds, and (2) those, perhaps more challenging and ambitious, that aim to permanently regenerate a fully functional epidermis, which requires targeting of epidermal stem cells. In both cases, the genetic variants underlying the different EB forms and factors, such as genetic background, modifier genes, comorbidities, and lifestyle, all of which impinge on EB genotype-phenotype correlation, need to be defined.I nherited epidermolysis bullosa (EB) is characterized by recurrent blistering of stratified epithelia. EB is caused by more than 1000 mutations in at least 16 structural genes encoding proteins forming hemidesmosomes and anchoring fibrils, which are essential for the integrity of the epidermal-dermal junction (Has et al. 2020a). Blisters arise spontaneously or upon minimal trauma as the result of the fragility of skin and mucous membranes. Common features of many EB forms include damage of ocular surface, upper airways, oral mucosa and gastrointestinal and renal systems, as well as hair, nail, and enamel defects (Bardhan et al. 2020;Has et al. 2020a). Incidence and prevalence of EB in the United States are 11.1 per one million people and 19.6 per one million live births, respectively. Similar values have been reported in Europe (Fine 2010;Has et al. 2020a). EB affects individuals from all ethnic origins regardless of gender and displays either dominant or recessive patterns of inheritance. EB phenotypes range from mild, localized blistering to massive blistering, erosions and chronic wounds. Severe EB forms can be early lethal and generalized EB frequently leads to aggressive squamous cell carcinoma (SCC) (Condorelli et al. 2019;Bardhan et al. 2020).

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Clonal analysis of human clonogenic keratinocytes

Cited by 11 publications

References 31 publications

Decoding the Human Epidermal Complexity at Single-Cell Resolution

Decoding the Human Epidermal Complexity at Single-Cell Resolution

Comparison between Cultivated Oral Mucosa and Ocular Surface Epithelia for COMET Patients Follow-Up

Stairways to Advanced Therapies for Epidermolysis Bullosa

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