Cultured Skin Fibroblasts Research Articles

Mutant WDR45 Leads to Altered Ferritinophagy and Ferroptosis in β-Propeller Protein-Associated Neurodegeneration.

Beta-propeller protein-associated neurodegeneration (BPAN) is a subtype of neurodegeneration with brain iron accumulation (NBIA) caused by loss-of-function variants in WDR45. The underlying mechanism of iron accumulation in WDR45 deficiency remains elusive. We established a primary skin fibroblast culture of a new BPAN patient with a missense variant p.(Asn61Lys) in WDR45 (NM_007075.3: c.183C>A). The female patient has generalized dystonia, anarthria, parkinsonism, spasticity, stereotypies, and a distinctive cranial MRI with generalized brain atrophy, predominantly of the cerebellum. For the functional characterization of this variant and to provide a molecular link of WDR45 and iron accumulation, we looked for disease- and variant-related changes in the patient’s fibroblasts by qPCR, immunoblotting and immunofluorescence comparing to three controls and a previously reported WDR45 patient. We demonstrated molecular changes in mutant cells comprising an impaired mitochondrial network, decreased levels of lysosomal proteins and enzymes, and altered autophagy, confirming the pathogenicity of the variant. Compared to increased levels of the ferritinophagy marker Nuclear Coactivator 4 (NCOA4) in control cells upon iron treatment, patients’ cells revealed unchanged NCOA4 protein levels, indicating disturbed ferritinophagy. Additionally, we observed abnormal protein levels of markers of the iron-dependent cell death ferroptosis in patients’ cells. Altogether, our data suggests that WDR45 deficiency affects ferritinophagy and ferroptosis, consequentially disturbing iron recycling.

Mitochondrial organization and structure are compromised in fibroblasts from patients with Huntington’s disease

ABSTRACT Huntington´s disease (HD) is a progressive neurodegenerative disease with onset in adulthood that leads to a complete disability and death in approximately 20 years after onset of symptoms. HD is caused by an expansion of a CAG triplet in the gene for huntingtin. Although the disease causes most damage to striatal neurons, other parts of the nervous system and many peripheral tissues are also markedly affected. Besides huntingtin malfunction, mitochondrial impairment has been previously described as an important player in HD. This study focuses on mitochondrial structure and function in cultivated skin fibroblasts from 10 HD patients to demonstrate mitochondrial impairment in extra-neuronal tissue. Mitochondrial structure, mitochondrial fission, and cristae organization were significantly disrupted and signs of elevated apoptosis were found. In accordance with structural changes, we also found indicators of functional alteration of mitochondria. Mitochondrial disturbances presented in fibroblasts from HD patients confirm that the energy metabolism damage in HD is not localized only to the central nervous system, but also may play role in the pathogenesis of HD in peripheral tissues. Skin fibroblasts can thus serve as a suitable cellular model to make insight into HD pathobiochemical processes and for the identification of possible targets for new therapies.

IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues.

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10−5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10−34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

Bioenergetic and Autophagic Characterization of Skin Fibroblasts from C9orf72 Patients.

The objective of this study is to describe the alterations occurring during the neurodegenerative process in skin fibroblast cultures from C9orf72 patients. We characterized the oxidative stress, autophagy flux, small ubiquitin-related protein SUMO2/3 levels as well as the mitochondrial function in skin fibroblast cultures from C9orf72 patients. All metabolic and bioenergetic findings were further correlated with gene expression data obtained from RNA sequencing analysis. Fibroblasts from C9orf72 patients showed a 30% reduced expression of C9orf72, ~3-fold increased levels of oxidative stress and impaired mitochondrial function obtained by measuring the enzymatic activities of mitochondrial respiratory chain complexes, specifically of complex III activity. Furthermore, the results also reveal that C9orf72 patients showed an accumulation of p62 protein levels, suggesting the alteration of the autophagy process, and significantly higher protein levels of SUMO2/3 (p = 0.03). Our results provide new data reinforcing that C9orf72 cells suffer from elevated oxidative damage to biomolecules and organelles and from increased protein loads, leading to insufficient autophagy and an increase in SUMOylation processes.

Germline CHEK2 and ATM Variants in Myeloid and Other Hematopoietic Malignancies

An intact DNA damage response is crucial to preventing cancer development, including in myeloid and lymphoid malignancies. Deficiencies in the homologous recombination (HR) pathway can lead to defective DNA damage responses, and this can occur through inherited germline mutations in HR pathway genes, such as CHEK2 and ATM. We now understand that germline mutations can be identified frequently (~ 5-10%) in patients with myeloid and lymphoid malignancies, and among the most common of these are CHEK2 and ATM. We review the role that deleterious germline CHEK2 and ATM variants play in the development of hematopoietic malignancies, and how this influences clinical practice, including cancer screening, hematopoietic stem cell transplantation, and therapy choice. In recent large cohorts of patients diagnosed with myeloid or lymphoid malignancies, deleterious germline loss of function variants in CHEK2 and ATM are among the most common identified. Germline CHEK2 variants predispose to a range of myeloid malignancies, most prominently myeloproliferative neoplasms and myelodysplastic syndromes (odds ratio range: 2.1-12.3), and chronic lymphocytic leukemia (odds ratio 14.83). Deleterious germline ATM variants have been shown to predispose to chronic lymphocytic leukemia (odds ratio range: 1.7-10.1), although additional studies are needed to demonstrate the risk they confer for myeloid malignancies. Early studies suggest there may also be associations between deleterious germline CHEK2 and ATM variants and development of clonal hematopoiesis. Identifying CHEK2 and ATM variants is crucial for the optimal management of patients and families affected by hematopoietic malignancies. OPENING CLINICAL CASE: "A 45year-old woman presents to your clinic with a history of triple-negative breast cancer diagnosed five years ago, treated with surgery, radiation, and chemotherapy. About six months ago, she developed cervical lymphadenopathy, and a biopsy demonstrated small lymphocytic leukemia. Peripheral blood shows a small population of lymphocytes with a chronic lymphocytic leukemia immunophenotype, and FISH demonstrates a complex karyotype: gain of one to two copies of IGH and FGFR3; gain of two copies of CDKN2C at 1p32.3; gain of two copies of CKS1B at 1q21; tetrasomy for chromosome 3; trisomy and tetrasomy for chromosome 7; tetrasomy for chromosome 9; tetrasomy for chromosome 12; gain of one to twocopies of ATM at 11q22.3; deletion of chromosome 13 deletion positive; gain of one to two copies of TP53 at 17p13.1). Given her history of two cancers, you arrange for germline genetic testing using DNA from cultured skin fibroblasts, which demonstrates pathogenic variants in ATM [c.1898 + 2T > G] and CHEK2 [p.T367Metfs]. Her family history is significant for multiple cancers. (Fig.1)." Fig. 1 Representative pedigree from a patient with germline pathogenic ATM and CHEK2 variants who was affected by early onset breast cancer and chronic lymphocytic leukemia. Arrow indicates proband. Colors indicate cancer type/disease: purple, breast cancer; blue, lymphoma; brown, melanoma; yellow, colon cancer; and green, autoimmune disease.

Pathogenic TP53 variant allele frequency across different somatic tissues in two patients with mosaic Li-Fraumeni.

10582 Background: The integration of germline genetic testing, frequently from blood, into routine oncological care has increased the frequency at which pathogenic TP53 mutations are found in a variant allele frequency (VAF) range suggestive of either mosaic Li-Fraumeni Syndrome or clonal hematopoiesis of indeterminate potential (CHIP). Mosaic LFS is a rare and poorly described phenomenon wherein a pathogenic TP53 mutation is dispersed in low levels through multiple somatic tissues after stochastically developing during early embryogenesis. In comparison, CHIP is restricted to the bone marrow niche. Germline mosaicism in Li-Fraumeni Syndrome (LFS) is inferred when genomic DNA from a second source recapitulates the exact same TP53 mutation. Given the challenges with sample acquisition, mosaicism has not been previously described in depth in the medical literature. Methods: Two pediatric patients with LFS-associated cancers (glioblastoma, osteosarcoma & ALL respectively) underwent clinical genetics evaluation and were found to have pathogenic TP53 mutations. Initial genetic testing was performed in a CLIA/CAP environment through a send out test or internally. A broad array of somatic tissues was collected at autopsy with subsequent DNA isolation and amplicon sequencing in triplicate to determine the mean VAF of the pathogenic TP53 allele. Results: For both patients, the initial germline test suggesting mosaic LFS had a VAF between 20-30%. Confirmatory intent fibroblast culture in Patient 1 was reported as negative by an external clinical lab and Patient 2’s VAF was much lower. The VAFs for the rest of the somatic tissues as well as in their corresponding cancers are reported below (Table) as means and standard error of the triplicate. Despite the negative fibroblast culture, Patient 1 did demonstrate mutant TP53 in other tissues, confirming mosaicism. Conclusions: The proportion of pathogenic alleles is highly heterogeneous amongst different somatic tissues in mosaic LFS patients. Although CHIP exclusion through germline testing of cultured skin fibroblasts is common, our patients’ skin samples were amongst the tissues with the lowest VAFs. Mosaicism should be highly suspected when tumor sequencing demonstrates an identical mutation to the putative germline mosaic one even with equivocal fibroblast culture results. [Table: see text]

Role of Cultured Skin Fibroblasts in Regenerative Dermatology.

The skin, as the largest organ, covers the entire outer part of the body, and since this organ is directly exposed to microbial, thermal, mechanical and chemical damage, it may be destroyed by factors such as acute trauma, chronic wounds or even surgical interventions. Cell therapy is one of the most important procedures to treat skin lesions. Fibroblasts are cells that are responsible for the synthesis of collagen, elastin, and the organization of extracellular matrix (ECM) components and have many vital functions in wound healing processes. Today, cultured autologous fibroblasts are used to treat wrinkles, scars, wounds and subcutaneous atrophy. The results of many studies have shown that fibroblasts can be effective and beneficial in the treatment of skin lesions. On the other hand, skin substitutes are used as a regenerative model to improve and regenerate the skin. The use of these alternatives, restorative medicine and therapeutic cells such as fibroblasts has tremendous potential in the treatment of skin diseases and can be a new window for the treatment of diseases with no definitive treatment. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description ofthese Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Impact of <i>Candida spp.</i> metabolites on human skin fibroblasts

Micromycetes spp. have been increasingly involved in the etiology of infectious diseases guiding to consider them not as important as bacterial and viral pathogens. Nowadays, a lot of severe forms of candidiasis are caused by C. auris, C. albicans, whereas C. glabrata and C. krusei are of similar importance. Members of these species were selected to investigate related metabolite action on human skin fibroblasts. Candida spp. being continuously found on the epithelium and mucosal membranes resulting in to sustained interaction between microbiota and human cells. Potential to produce metabolites containing pathogenicity factors is one of the crucial events for transition to invasive candidiasis, wherein human epithelial cells build up the front line of defense barrier preventing Candida spp. invasion into deeper host tissues. The study was aimed at assessing data on metabolite effects derived from epidemiologically relevant Candida spp. on primary human skin fibroblast culture in vitro. In particular, there were analyzed Candida spp. metabolites acting on fibroblast monolayer integrity and viability in cell suspension. It was found that Candida spp. metabolites might directly cause fibroblast death so that biocidal activity was exhibited as a strain-specific feature. A direct biocidity against dermal cells was more typical for strains C. glabrata и C. krusei, less pronounced for C. albicans and very weak for C. auris. In addition, a mechanism for secretory product-related biocidal activity derived from various Candida spp. on dermal fibroblasts in vitro revealed that it resulted in fibroblasts death 1 hour after exposure that peaked at 3 hrs. Cell death was equally proceeded via apoptosis and necrosis. Of note, biocidal effect of fungal metabolites showed no correlation with Candida-related potential to cleave intercellular junctions. It was found that C. auris metabolites showing weak biocidity against some fibroblasts simultaneously resulted in more marked disruption of cell monolayer compared to other Candida spp. Perhaps, it is just a feature of C. auris that might account for its higher invasiveness potential allowing to destroy tight human tissues more effectively compared to other Candida spp.

Feasibility and limitations of cultured skin fibroblasts for germline genetic testing in hematologic disorders.

To avoid acquired variants found in the blood, cultured skin fibroblasts are a recommended DNA source for germline genetic testing in patients with hematologic disorders, but data are lacking regarding practicality and limitations. We conducted a retrospective cohort study of 350 subjects with hematologic disorders who underwent skin fibroblast culture for germline genetic testing. We analyzed next‐generation sequencing data from the targeted capture of 144 inherited cancer and bonemarrow failure genes to identify variants at heterozygous and subclonal variant allele frequencies. Sixteen (5%) biopsies failed to culture. Culture failure was more likely in samples with delays in culture initiation (OR = 4.3; p < 0.01) or a pathogenic variant in a telomere gene (OR = 42.6; p < 0.01). Median culture time was 28 days (IQR 22−29 days). Culture time was longer for subjects with prior allogeneic stem cell transplantation (+10.7%; p = 0.02) and shorter in subjects with a heterozygous pathogenic variant (−11.9%; p < 0.01), larger biopsy size (−10.6%; p < 0.01), or lymphoid malignancy (−8.4%; p < 0.01). Subclonal variants were identified in 10 (4%) and confirmed in five (56%) of eight with alternate samples available. Subclonal and discordant variants illustrate that germline testing from cultured skin fibroblasts requires phenotypic correlation and, in rare cases, follow‐up studies for optimal interpretation.

Biomimetic macroporous hydrogel with a triple-network structure for full-thickness skin regeneration

Full-thickness skin repair is still a challenge in clinical practice because it is extremely difficult for dermal reconstruction and calls for a promising strategy to achieve quick sutureless closure and high-quality dermal restoration for large acute skin defects. Inspired by the unique composition, mechanical properties and microscopic architecture of skin tissue, a novel biomimetic hydrogel with a triple-network structure that mimics the extracellular matrix (ECM) composition has been reported. A composite biomimetic hydrogel was prepared using ECM-derived biopolymers, gelatin methacryloyl (GelMA) and N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2‑methoxy-5-nitro-sophenoxy) butanamide (NB)-linked sodium alginate (Alg-NB) and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). This biomimetic hydrogel can undergo rapid gelling under UV irradiation (approximately 3 s), has strong mechanical properties (mechanical strength ≈530 kPa) with UV-ionic-crosslinking, and shows strong adhesion to wet tissue. In vitro two-dimensional culture of human skin fibroblasts (HSFs) confirmed that the biomimetic hydrogel has excellent biocompatibility, and HSFs on the surface grow into cell clusters because of the macroporous structures of the hydrogel (pore diameter ≈160 μm). The subcutaneous implantation of the biomimetic hydrogel in Sprague-Dawley (SD) rats confirmed its biocompatibility and low biodegradation in vivo. Moreover, in an SD rat full-thickness skin defect model, this engineered biomimetic hydrogel not only could be used for sutureless wound closure strategies but also could accelerate the reconstitution of dermal tissue with skin appendages. This study provides an effective strategy of quick sutureless wound closure and highly efficient repair for full-thickness skin defects and shows enormous potential for tissue regeneration in clinical applications.

Genotype-phenotype correlations in CPT1A deficiency detected by newborn screening in Pacific populations.

Carnitine palmitoyltransferase 1A (CPT1A) deficiency is a long chain fatty acid oxidation disorder, typically presenting with hypoketotic hypoglycaemia and liver dysfunction during fasting and intercurrent illness. Classical CPT1A deficiency is a rare disease, although a milder ‘Arctic variant' (p.P479L) is common in the Inuit population. Since the introduction of expanded metabolic screening (EMS), the newborn screening programmes of Hawai'i and New Zealand (NZ) have detected a significant increase in the incidence of CPT1A deficiency. We report 22 individuals of Micronesian descent (12 in NZ and 10 in Hawai'i), homozygous for a CPT1A c.100T>C (p.S34P) variant detected by EMS or ascertained following diagnosis of a family member. No individuals with the Micronesian variant presented clinically with metabolic decompensation prior to diagnosis or during follow‐up. Three asymptomatic homozygous adults were detected following the diagnosis of their children by EMS. CPT1A activity in cultured skin fibroblasts showed residual enzyme activity of 26% of normal controls. Secondly, we report three individuals from two unrelated Niuean families who presented clinically with symptoms of classic CPT1A deficiency, prior to the introduction of EMS. All were found to be homozygous for a CPT1A c.2122A>C (p.S708R) variant. CPT1A activity in fibroblasts of all three individuals was severely reduced at 4% of normal controls. Migration pressure, in part due to climate change may lead to increased frequency of presentation of Pacific peoples to regional metabolic services around the world. Knowledge of genotype–phenotype correlations in these populations will therefore inform counselling and treatment of those detected by newborn screening.

Technical Aspects of Coenzyme Q10 Analysis: Validation of a New HPLC-ED Method.

The biochemical measurement of the CoQ status in different tissues can be performed using HPLC with electrochemical detection (ED). Because the production of the electrochemical cells used with the Coulochem series detectors was discontinued, we aimed to standardize a new HPLC-ED method with new equipment. We report all technical aspects, troubleshooting and its performance in different biological samples, including plasma, skeletal muscle homogenates, urine and cultured skin fibroblasts. Analytical variables (intra- and inter-assay precision, linearity, analytical measurement range, limit of quantification, limit of detection and accuracy) were validated in calibrators and plasma samples and displayed adequate results. The comparison of the results of a new ERNDIM external quality control (EQC) scheme for the plasma CoQ determination between HPLC-ED (Lab 1) and LC-MS/MS (Lab 2) methods shows that the results of the latter were slightly higher in most cases, although a good consistency was generally observed. In conclusion, the new method reported here showed a good analytical performance. The global quality of the EQC scheme results among different participants can be improved with the contribution of more laboratories.

ЭФФЕКТИВНОСТЬ ЛЕЧЕНИЯ ОЖОГОВОЙ ТРАВМЫ НА МОДЕЛИ СПЛОШНОГО И МОЗАИЧНОГО ОЖОГОВ У ЖИВОТНЫХ С ИСПОЛЬЗОВАНИЕМ ТКАНЕВОГО ЭКВИВАЛЕНТА КОЖИ

The effectiveness of the treatment of thermal burns in experimental animals using the developed skin tissue equivalent based on cultured skin fibroblasts and keratinocytes (TES) was studied. The terms of wound healing in experimental and control groups of animals with thermal burns IIIAB­IV degree were studied. An analysis was carried out using morphological methods for studying sections of biopsy specimens of burn wounds of the skin of animals at various periods of healing. The stimulating effect of TES on regenerative processes in wounds was revealed. When using TES in animals with solid and mosaic burns, there was a shortening of the wound healing time by 1.64 times compared with the control group of untreated animals.

Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE.

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.

Stimulatory effects of collagen production induced by coenzyme Q&lt;sub&gt;10&lt;/sub&gt; in cultured skin fibroblasts

Coenzyme Q10 (CoQ10) is a well-known antioxidant and serves as an essential carrier for electron transport and proton translocation in the mitochondrial respiratory chain. CoQ10 has been widely commercially available in Japan as a dietary and health supple­ment since 2001 and it is used for the prevention of lifestyle-related diseases induced by aging. Recently, it was stated that for Japan, which is facing an aging society, CoQ10 has been used in many skincare products. However, the physiological actions of CoQ10 in skin fibroblasts are not fully understood. In this study, we examined the effect of CoQ10 on cultured human skin fibroblast. In this study, CoQ10 treatment increased intracellular CoQ10 level and promoted proliferation of fibroblasts. In addition, CoQ10 increased mRNA expression of type I, IV, VII collagen, elastin, and HSP47, whereas CoQ10 has little effect on mRNA of type II and VIII MMP. These results suggested that CoQ10 has the efficacy that it increases collagen production in skin, thereby there is possible of the anti-aging by CoQ10 in Japan which reached an aging society, so that it might be based on new physiological function by CoQ10.

Disease Characteristics of AML Patients with Germline DDX41 Variants

Disease Characteristics of AML Patients with Germline DDX41 Variants

Effects of E407a on the viability, metabolic and functional activity of dermal fibroblasts

Aim. To evaluate the effects of semi-refined carrageenan (E407a) on the viability, metabolic and functional activity of skin fibroblasts in vitro. Materials and methods. Various concentrations of E407a (0 – 10 mg/ml) were incubated for 24 h with skin fibroblast cultures isolated from rat embryos. The viability of cells exposed to semi-refined carrageenan was assessed by the neutral red uptake assay. MTT test was selected to estimate the metabolic activity of cells. In addition, scratch assay was used to determine the functional activity of dermal fibroblasts. Results. Direct exposure to semi-refined carrageenan did not affect the viability of rat dermal fibroblasts, evidenced by the results of MTT and neutral red uptake assays. However, experimental data of MTT and scratch assays revealed that high concentrations of this food additive increased the metabolic activity of fibroblasts and diminished the motility of these cells, suggesting that E407a is cytotoxic at high concentrations. Conclusion. Food-grade semi-refined carrageenan is cytotoxic towards skin fibroblasts at high concentrations.

Signaling Molecules of Human Skin Cells as the Targets for Injection Cosmetology.

IntroductionSkin aging is a natural process that cannot be stopped. However, there are many ways to help attenuate premature aging of the skin and reduce the signs that have already appeared. One of them is the subcutaneous administration of preparations containing a combination of hyaluronic acid, active amino acids, and peptides providing an anti-aging clinical effect. The purpose of this research is to study in vitro new signaling molecules with the anti-aging effects and influence of hyaluronic acid fillers on its expression.MethodsThe study was conducted using cell cultures of human facial skin: 1) mixed culture of human facial skin keratinocytes and fibroblasts, and 2) culture of human facial skin keratinocytes enriched with Merkel cells. Immunocytochemistry, confocal microscopy and Western blot were used to identify markers of aging.ResultsHA-Y and HA-S activated the expression of Klotho in the case of aging mixed culture of human skin keratinocytes and Merkel cells. The increase in expression of MTH-1 with aging of cultures provides evidence of activating defense mechanisms against reactive oxygen species that are accumulating with aging, under the action of HA-S and HA-Y. There was a statistically valid increase in the area of expression of melatonin receptor 1A and 1B markers when adding both HA-S and HA-Y to cultured cells.ConclusionThis investigation showed that the studied fillers have biological effects, testifying the stimulation of reparative processes in the skin under their control.

Method Optimization of Skin Biopsy-Derived Fibroblast Culture for Reprogramming Into Induced Pluripotent Stem Cells.

Background: Fibroblasts can be isolated from skin biopsies using a chemical dissociation, a physical dissociation, or a combination of both techniques. They can be reprogrammed into induced pluripotent stem cells (iPSCs) through the introduction of defined sets of key transcription factors. This study aimed to identify the optimal protocol for skin biopsy dissociation, fibroblast culture, and fibroblast cryopreservation in the scope of reprogramming into iPSCs and in the context of biobank accreditation. Methods: First, four dissociation techniques typically used in the laboratory (explant based, enzymatic, and/or mechanical) and two cryopreservation media containing 10% dimethyl sulfoxide, either commercial or homemade, were evaluated in terms of post-thaw recovery, viability, growth curves, and karyotyping analyses of the fibroblasts. Next, the clones reprogrammed from the fibroblasts isolated with the two optimal dissociation methods and cryopreservation media were further assessed by reprogramming quality before cryopreservation and post-thaw pluripotency comparison. Results: Fibroblasts isolated from skin biopsies using an explant-based or enzymatic dissociation method showed higher viability, higher proliferative potential, and higher genome stability post-thaw compared to the other dissociation techniques. Fibroblasts obtained by the explant-based dissociation technique showed a slightly higher reprogramming quality. The iPSC reprogrammed from explant-based dissociated fibroblasts showed successful recovery of iPSC clones. No difference between the two cryopreservation media was detected for the tested endpoints, with the exception of a higher visual count of colonies at the end of the reprogramming for the explant-based dissociation method. Conclusions: This article presents a formal method optimization for biospecimen processing in the context of accreditation in laboratories and biobanks. We validated skin biopsy-derived fibroblast isolation, culture, and cryopreservation for downstream mRNA reprogramming into iPSCs. The explant-based dissociation technique and homemade medium are selected as optimal to isolate and cryopreserve fibroblasts from skin biopsies in the scope of reprogramming into iPSCs.

Fetal brain arrest broadens the spectrum of WDR81-related developmental brain malformations.

Fetal brain arrest is an extremely rare genetic disorder that was described in few patients and encompasses very unique findings of underdeveloped cerebral hemispheres in association with collapsed skull bones. Based on the recurrence among sibs, an autosomal recessive mode of inheritance was proposed; however, no causative gene was identified so far. Here, we report the identification of biallelic variants in the WDR81 gene in two unrelated families (4 patients) with fetal brain arrest including the originally described family and an additional new family. Two homozygous variants were identified: a new missense (c.1157T > C, p.Val386Ala) and a previously described frameshift variant, c.4668_4669delAG (p.Gly1557AspfsTer16). We assessed the expression of WDR81 at the protein level by western blot analysis using primary skin fibroblast cultures established from the patient with the missense variant and noticed that WDR81 expression was significantly reduced in comparison to normal control confirming the pathogenicity of this variant. Our findings confirm the involvement of WDR81 in the pathogenesis of fetal brain arrest syndrome and suggest that fetal brain arrest represents the severe end of the spectrum phenotypes caused by pathogenic variants in WDR81. In addition, we reviewed the clinical and molecular data on WDR81-related disorders and phenotype/genotype correlations.