KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes.

Expanded autologous skin keratinocytes are currently used in cutaneous cell therapy, and embryonic-stem-cell-derived keratinocytes could become a complementary alternative. Regardless of keratinocyte provenance, for efficient therapy it is necessary to preserve immature keratinocyte precursors during cell expansion and graft processing. Here, we show that stable and transient downregulation of the transcription factor Krüppel-like factor 4 (KLF4) in keratinocyte precursors from adult skin, using anti-KLF4 RNA interference or kenpaullone, promotes keratinocyte immaturity and keratinocyte self-renewal in vitro, and enhances the capacity for epidermal regeneration in mice.

Single thrombopoietin dose alleviates hematopoietic stem cells intrinsic short- and long-term ionizing radiation damage. In vivo identification of anatomical cell expansion sites.

Hematopoietic stem cells (HSC) are essential for maintaining the integrity of complex and long-lived organisms. HSC, which are self-renewing, reconstitute the hematopoietic system through out life and facilitate long-term repopulation of myeloablated recipients. We have previously demonstrated that when mice are exposed to sublethal doses of ionizing radiation, subsets of the stem/progenitor compartment are affected.

Monitoring the cycling activity of cultured human keratinocytes using a CFSE-based dye tracking approach.

The development of methods and tools suitable for functional analysis of keratinocytes placed in an in vitro context is of great importance for characterizing properties associated with their normal state, for detecting abnormalities related to pathological states, or for studying the effects of extrinsic factors. In the present chapter, we describe the use of the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) to monitor cell division in mass cultures of normal human keratinocytes. We detail the preparation of CFSE-labeled keratinocyte samples and the identification by flow cytometry of cell subpopulations exhibiting different cycling rates in a mitogenic culture context.

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation.

Purpose: Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair.

Cellular adhesion on collagen: a simple method to select human basal keratinocytes which preserves their high growth capacity.

The regenerative capacity of human interfollicular epidermis is closely linked to the potential of immature keratinocytes present within its basal layer. The availability of selection methods and culture systems allowing precise assessment of basal keratinocyte characteristics is critical for increasing our knowledge of this cellular compartment. This report presents a multi-parametric comparative study of basal keratinocytes selected according to two different principles: 1) high adhesion capacity on a type-I collagen-coated substrate [Adh⁺⁺⁺], 2) high cell-surface expression of α6-integrin [Itg-α6 (high)].

Transcriptome characterization uncovers the molecular response of hematopoietic cells to ionizing radiation.

Ionizing radiation causes rapid and acute suppression of hematopoietic cells that manifests as the hematopoietic syndrome. However, the roles of molecules and regulatory pathways induced in vivo by irradiation of different hematopoietic cells have not been completely elaborated. Using a strategy that combined different microarray bioinformatics tools, we identified gene networks that might be involved in the early response of hematopoietic cells radiation response in vivo.

Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells.

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference.

Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes.

The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi-parallel clonal cultures of basal keratinocytes.

Functional investigations of keratinocyte stem cells and progenitors at a single-cell level using multiparallel clonal microcultures.

The basal layer of human interfollicular epidermis is thought to contain a minor compartment of quiescent or slowly cycling epithelial stem cells. These primitive keratinocytes give rise to the progenitors, which are the proliferating keratinocytes and which can be defined as early to late progenitors, according to their differentiation status. Because of the intrinsic heterogeneity of the basal layer, the development of new methods suitable for functional analysis of basal keratinocytes directly isolated from skin samples is greatly needed.

A Myc-regulated transcriptional network controls B-cell fate in response to BCR triggering.

Background: The B cell antigen receptor (BCR) is a signaling complex that mediates the differentiation of stage-specific cell fate decisions in B lymphocytes. While several studies have shown differences in signal transduction components as being key to contrasting phenotypic outcomes, little is known about the differential BCR-triggered gene transcription downstream of the signaling cascades.

Results: Here we define the transcriptional changes that underlie BCR-induced apoptosis and proliferation of immature and mature B cells, respectively.

Phenotypic and functional changes induced in hematopoietic stem/progenitor cells after gamma-ray radiation exposure.

Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period.

Sensing radiosensitivity of human epidermal stem cells.

Purpose: Radiosensitivity of stem cells is a matter of debate. For mouse somatic stem cells, both radiosensitive and radioresistant stem cells have been described. By contrast, the response of human stem cells to radiation has been poorly studied.

Comparison of gene expression pattern in SP cell populations from four tissues to define common "stemness functions".

The goal of our study was to identify a subset of genes commonly expressed in Side Populations (SP), isolated by Hoechst staining followed by flow cytometry, from adult mouse bone marrow, male adult germinal cells, muscle primary culture, and mesenchymal cells. These SP cells have been proposed to be a "stem-like" population and are used here as a "model" that may reveal mechanisms which would be relevant for a better understanding of stem cell properties. Transcriptional profiles for SP and the more differentiated non-SP cells isolated from the four tissues were compared by hybridization on microarray using a common external reference.

Human side population keratinocytes exhibit long-term proliferative potential and a specific gene expression profile and can form a pluristratified epidermis.

The aim of the present study was to characterize human side population (SP) epidermal keratinocytes isolated from primary cell cultures. For that purpose, keratinocytes were isolated from normal adult breast skin samples and the Hoechst 33342 exclusion assay described for hematopoietic cells was adapted to keratinocytes. Three types of keratinocytes were studied: the SP, the main population (MP), and the unsorted initial population.

CD98, a novel marker of transient amplifying human keratinocytes.

Identification of plasma membrane markers of basal keratinocytes is essential for sorting basal cells and, subsequently, adult epidermal stem cells. In this study, we isolated caveolin-1-enriched microdomains from human HaCaT keratinocytes and identified proteins representing potential cell surface markers of the epidermis by a proteomic approach. The purification of this caveolae domain allowed us to characterize 53 proteins of which 26% were transmembrane and 32% associated-membrane proteins.

Novel pathway for megakaryocyte production after in vivo conditional eradication of integrin alphaIIb-expressing cells.

Our knowledge of the molecular mechanisms that regulate hematopoiesis in physiologic and pathologic conditions is limited. Using a molecular approach based on cDNA microarrays, we demonstrated the emergence of an alternative pathway for mature bone marrow cell recovery after the programmed and reversible eradication of CD41+ cells in transgenic mice expressing a conditional toxigene targeted by the platelet alphaIIb promoter. The expression profile of the newly produced CD41+ cells showed high levels of transcripts encoding Ezh2, TdT, Rag2, and various immunoglobulin (Ig) heavy chains.

(alpha)IIb Integrin, a novel marker for hemopoietic progenitor cells.

Integrin (alpha)IIb(beta)3 (abbreviated as (alpha)IIb), also known as GPIIb-IIIa or CD41/CD61, is a cell adhesion molecule expressed on cells belonging to the megakaryocytic lineage. Aiming to identify new markers of hemopoietic progenitor cells (HPC), we undertook a developmental study of this molecule since it remains controversial if this integrin is expressed by various progenitors. We reported the expression pattern of two integrins, in both of which the beta3 chain is present, respectively associated with alphaV and alpha IIb in the chick embryo.

The instability of the neural crest phenotypes: Schwann cells can differentiate into myofibroblasts.

In the vertebrate embryo, the neural crest cells (NCCs) that migrate out from the neural primordium yield multiple phenotypes, including melanocytes, peripheral neurones and glia and, in the head, cartilage, bone, connective cells and myofibroblasts / vascular smooth muscle cells (SMCs). The differentiation of pluripotent NCCs is mainly directed by local growth factors. Even at postmigratory stages, NC-derived cells exhibit some fate plasticity.

Expression profiling of genes and proteins in HaCaT keratinocytes: proliferating versus differentiated state.

The knowledge of the mechanism of keratinocyte differentiation in culture is still uncompleted. The emergence of new technologies, such as cDNA microarrays or 2D electrophoresis followed by mass spectrometry analysis, has allowed the identification of genes and proteins expressed in biological processes in keratinocytes. Here, we report a genome wide analysis of proliferating versus differentiated human HaCaT keratinocytes.

Reversal of developmental restrictions in neural crest lineages: transition from Schwann cells to glial-melanocytic precursors in vitro.

In vertebrate embryos, diversification of the lineages arising from the neural crest (NC) is controlled to a large extent by environmental factors. In previous work, we showed that endothelin 3 (ET3) peptide favors the development of glial and melanocytic NC precursors in vitro. This factor is also capable of inducing proliferation of cultured epidermal pigment cells and their conversion to glia.

Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor.

Functional signaling of endothelin 3 (ET3) and its receptor B (ETRB) has been shown to be required for the development of neural crest (NC)-derived pigment cells in mouse, but the precise role of ET3 is not completely understood. Using the avian embryo as a model, we previously reported that ET3 promotes the survival and proliferation of unipotent melanocyte and bipotent glia-melanocyte precursors in trunk NC cultures. Here we investigated whether, at later stages, embryonic pigment cells respond to ET3.

Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells.

The fibrinogen receptor GPIIb-IIIa integrin is known to be expressed on cells of the megakaryocytic lineage, but its presence on hematopoietic progenitors has been a controversial issue. To resolve this ambiguity unequivocally, we performed clonogenic assays and intrathymic cell-transfer experiments in congenic animals. As the ontogeny of the avian hematopoietic system is well documented, we used this experimental model to trace GPIIb-IIIa expression during embryogenesis.

Ligand-dependent development of the endothelial and hemopoietic lineages from embryonic mesodermal cells expressing vascular endothelial growth factor receptor 2.

The existence of a common precursor for endothelial and hemopoietic cells, termed the hemangioblast, has been postulated since the beginning of the century. Recently, deletion of the endothelial-specific vascular endothelial growth factor receptor 2 (VEGFR2) by gene targeting has shown that both endothelial and hemopoietic cells are absent in homozygous null mice. This observation suggested that VEGFR2 could be expressed by the hemangioblast and essential for its further differentiation along both lineages.