Effects of TP63 mutations on keratinocyte adhesion and migration.

The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced AEC iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.

Effects of TP63 Mutations on Keratinocyte Adhesion and Migration.

The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.

Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes.

Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create "designer" iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC-derived keratinocytes (iPSC-K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™ 400 6-well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells.

Safety and immune regulatory properties of canine induced pluripotent stem cell-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) exhibit broad immune modulatory activity in vivo and can suppress T cell proliferation and dendritic cell activation in vitro. Currently, most MSC for clinical usage are derived from younger donors, due to ease of procurement and to the superior immune modulatory activity. However, the use of MSC from multiple unrelated donors makes it difficult to standardize study results and compare outcomes between different clinical trials. One solution is the use of MSC derived from induced pluripotent stem cells (iPSC); as iPSC-derived MSC have nearly unlimited proliferative potential and exhibit in vitro phenotypic stability. Given the value of dogs as a spontaneous disease model for pre-clinical evaluation of stem cell therapeutics, we investigated the functional properties of canine iPSC-derived MSC (iMSC), including immune modulatory properties and potential for teratoma formation. We found that canine iMSC downregulated expression of pluripotency genes and appeared morphologically similar to conventional MSC. Importantly, iMSC retained a stable phenotype after multiple passages, did not form teratomas in immune deficient mice, and did not induce tumor formation in dogs following systemic injection. We concluded therefore that iMSC were phenotypically stable, immunologically potent, safe with respect to tumor formation, and represented an important new source of cells for therapeutic modulation of inflammatory disorders.

Hematopoietic Development of ES Cells in Culture.

Under appropriate culture conditions, ES cells will spontaneously differentiate and generate colonies known as embryoid bodies (EBs) that contain precursors of multiple lineages, including those of the hematopoietic system (1-7). Previous studies have demonstrated that the molecular events leading to hematopoietic commitment, as well as the kinetics of lineage development within the EBs, parallel that found in the normal mouse embryo (5). More recent studies (8-11) have supported these earlier findings and have provided evidence that hematopoietic development within EBs can be divided into the following distinct stages: hemangioblast, primitive and early definitive, and multilineage definitive. These stages most closely correspond to the preblood island, the early-mid yolk sac, and the late yolk sac-early fetal-liver hematopoietic programs within the mouse embryo.

MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF-2 in fibroblasts.

Mitogen-activated protein kinases (MAPKs) are regulated by MAPK kinases (MKKs), which are in turn regulated by MKK kinases (MKKKs). While a single MKKK can regulate several different MAPK family members, and several MKKKs can often activate the same MAPK, emerging evidence indicates a unique role for individual MKKKs in acting as signaling nodes to coordinately activate different subsets of MAPKs in response to specific cellular stimuli. Thus, while there is much apparent overlap in MAPK regulation by different MKKKs, each MKKK serves a specific purpose in regulation of unique cellular functions. The purpose of this study was to define the specific role of MEKK2, an MKKK, in MAPK regulation and cell function. MEKK2 coordinately activates the ERK5 and JNK pathways. Targeted disruption of MEKK2 expression causes loss of ERK5 and JNK activation in response to FGF-2 in mouse embryonic fibroblasts (MEFs). FGF-2 receptor signaling requires MEKK2 for induction of mRNA for c-Jun, Fra-1, and Fra-2, components of the AP-1 transcription complex. In FGF-2-stimulated MEKK2-/- fibroblasts, c-Jun phosphorylation is inhibited, consistent with a loss of JNK activation. Thus, MEKK2 regulates AP-1 activity at two levels, by regulating both expression of AP-1 components and c-Jun N-terminal phosphorylation. One function of the AP-1 transcription complex is to regulate cytokine gene expression. Expression of IL-1alpha, IL-1beta, IL-6, and TNFalpha is inhibited in MEKK2-/- fibroblasts. Bacterial lipopolysaccharide (LPS) and TNFalpha neither activate ERK5 nor require MEKK2 for JNK activation, demonstrating specificity of MEKK2 in FGF-2 receptor signaling and control of cytokine gene expression.

The in vitro production and characterization of neutrophils from embryonic stem cells.

An embryonic stem (ES) cell/OP9 coculture system for the effective production of functional neutrophils is described. A 3-step differentiation strategy was developed that uses liquid culture, enabling reliable and abundant production of neutrophils at high purity without the need of sorting for isolation of mature neutrophils. Use of the OP9 stromal cell line significantly enhances the number, percentage, and duration of differentiated neutrophils produced from embryonic stem cells. Effective and sustained differentiation of ES cells into neutrophils provides a useful model system for studying neutrophil differentiation and function and the factors that regulate them. Morphologic and functional evaluation of these ES-derived neutrophils indicates that large numbers of mature neutrophils can be produced from pluripotent ES cells in vitro. Specifically, their morphology, ability to produce superoxides, flux calcium, undergo chemotaxis in response to macrophage inflammatory protein 2 (MIP-2), stain for the granulocyte-specific marker-specific chloroacetate esterase, and contain the neutrophil-specific markers Gr-1 and the mouse neutrophil-specific antigen indicates that they are comparable with purified mouse bone marrow neutrophils. They also express gelatinase and lactoferrin granule proteins. During the differentiation of these ES-derived neutrophils, regional areas of neutrophil production can be identified that have been designated as neutrophil generating regions (NGRs).

MEF2C regulates c-Jun but not TNF-alpha gene expression in stimulated mast cells.

Mitogen-activated protein kinase (MAPK) cascades play essential roles in the transduction of extracellular signals to cytoplasmic and nuclear effectors. The MAPK kinase kinase MEKK2 is essential for activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 5 (ERK5). These pathways are important for expression of specific cytokine genes in mast cells following cross-linking of the high-affinity IgE receptor (FcepsilonRI). A consequence of ERK5 activation is activation of the transcriptional factor myocyte enhancing factor-2C (MEF2C), leading to increased c-Jun expression. We have investigated the role of MEF2C activation in mast cells and demonstrated that it requires sequential activation of the signaling cascade of MEKK2-MEK5-ERK5. Following phosphorylation of MEF2C, activated MEF2C regulates transcription of c-Jun but not TNF-alpha. Inhibition of ERK5, MEK5 activation or activation of MEKK2-deficient mast cells was associated with inhibition of MEF2C phosphorylation and a decrease in c-Jun expression. Thus, these data define an activation module, MEKK2-MEK5-ERK5-MEF2C in the transcriptional activation of c-Jun in mast cells following FcepsilonRI cross-linking. These results demonstrate the novel and important, MEKK2-dependent role of MEF2C in induction of c-Jun expression in mast cells activated through FcepsilonRI, a pathway distinct from that involving MEKK2-MEK5-ERK5 in the regulation of mast cell cytokine production.