Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Mpox virus infection and drug treatment modelled in human skin organoids.

Mpox virus (MPXV) primarily infects human skin to cause lesions. Currently, robust models that recapitulate skin infection by MPXV are lacking. Here we demonstrate that human induced pluripotent stem cell-derived skin organoids are susceptible to MPXV infection and support infectious virus production. Keratinocytes, the predominant cell type of the skin epithelium, effectively support MPXV infection. Using transmission electron microscopy, we visualized the four stages of intracellular virus particle assembly: crescent formation, immature virions, mature virions and wrapped virions. Transcriptional analysis showed that MPXV infection rewires the host transcriptome and triggers abundant expression of viral transcripts. Early treatment with the antiviral drug tecovirimat effectively inhibits infectious virus production and prevents host transcriptome rewiring. Delayed treatment with tecovirimat also inhibits infectious MPXV particle production, albeit to a lesser extent. This study establishes human skin organoids as a robust experimental model for studying MPXV infection, mapping virus-host interactions and testing therapeutics.

Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids.

Human induced pluripotent stem cell (hiPSC)-derived hair-bearing skin organoids offer exciting new possibilities for modeling diseases like epidermolysis bullosa (EB). These inherited diseases affect 1 in 30,000 people worldwide and result from perturbed expression and/or structure of components of the epidermal-dermal junction (EDJ). To establish whether hiPSC-derived skin organoids might be able to capture salient features of EB, it is thus important to characterize their EDJ. Here, we report successful generation of hair-bearing skin organoids from two hiPSC lines that exhibited fully stratified interfollicular epidermis. Using immunofluorescence and electron microscopy, we showed that basal keratinocytes in organoids adhere to laminin-332 and type IV collagen-rich basement membrane via type I hemidesmosomes and integrin ß1-based adhesion complexes. Importantly, we demonstrated that EDJs in organoids are almost devoid of type VII collagen, a fibril that mediates anchorage of the epidermis to dermis. This should be considered when using skin organoids for EB modeling.

Generation and genetic repair of two human induced pluripotent cell lines from patients with Epidermolysis Bullosa simplex and dilated cardiomyopathy associated with a heterozygous mutation in the translation initiation codon of KLHL24.

Fibroblasts from two patients carrying a heterozygous mutation in the translation initiation codon (c.2 T > G) of the kelch-like protein 24 (KLHL24) gene were used to generate human induced pluripotent stem cells (hiPSCs), using non-integrating Sendai virus to deliver reprogramming factors. CRISPR-Cas9 editing was used for genetic correction of the mutation in the patient-hiPSCs. The top-predicted off-target sites were not altered. Patient and isogenic hiPSCs showed typical morphology, expressed pluripotency-associated markers, had the capacity for in vitro differentiation into the three germ layers and displayed a normal karyotype. These isogenic pairs will enable in vitro modelling of KLHL24-associated heart and skin conditions.

Lymphoblast-derived hiPS cell lines generated from four individuals of a family of genetically unrelated parents and their female monozygotic twins.

Lymphoblast cells from four individuals of a family of two genetically unrelated parents and their monozygotic twins were used to generate integration-free human induced pluripotent stem cells (hiPSCs). Reprogramming factors were delivered by co-electroporation of three episomal-based plasmids expressing OCT3/4, SOX2, KLF4, L-MYC and LIN28. The hiPSCs showed a normal karyotype, expressed pluripotency-associated markers, displayed the capacity for in vitro differentiation into the three germ layers and were Epstein Barr virus-free. These hiPSC lines offer the possibility to compare genetically unrelated and genetically identical tissues from different individuals and to study genotype-specific effects, which are particularly relevant for toxicology testing.

Deciphering the Mammary Stem Cell Niche: A Role for Laminin-Binding Integrins.

Integrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.

Decreased Expression of Vascular Endothelial Growth Factor Receptor 1 Contributes to the Pathogenesis of Hereditary Hemorrhagic Telangiectasia Type 2.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. METHODS: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. RESULTS: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. CONCLUSIONS: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.

Contractility Assay for Established Myoepithelial Cell Lines.

The capacity of mammary myoepithelial cells to contract in response to suckling stimuli is essential for lactation. We describe here a protocol for studying the contractile activity of myoepithelial cells in vitro. This protocol includes the establishment of stable myoepithelial cell lines from mouse mammary glands and quantitative evaluation of the contraction and subsequent relaxation of cultured myoepithelial cells in response to oxytocin. It can be used for analyses of mouse mutants with gene deletions or overexpression altering myoepithelial cell function.

Delayed Healing of Sickle Cell Ulcers Is due to Impaired Angiogenesis and CXCL12 Secretion in Skin Wounds.

Leg ulcers are a major complication of sickle cell disease that occur in 2.5-40% of patients. Leg ulcers are responsible for frequent complications because they are often long-lasting and are highly resistant to therapy. Although their occurrence is associated with hyperhemolysis, the mechanisms underlying sickle cell ulcers remain poorly understood. In this study, we show that skin wound healing is severely altered in old SAD sickle cell mice but is normal in young animals, consistent with reports in humans. Alterations of wound healing were associated with impaired blood and lymphatic angiogenesis in the wound beds and poor endothelial progenitor cell mobilization from the bone marrow. CXCL12 secretion by keratinocytes and inflammatory cells was low in the wounds of SAD mice. Local therapy with endothelial progenitor cells or recombinant CXCL12 injections restored wound angiogenesis and rescued the healing defect together with mobilization of circulating endothelial progenitor cells. To our knowledge, this is a previously unreported study of the cellular and molecular mechanisms of sickle cell ulcers in a murine model that provides promising therapeutic perspectives for clinical trials.

The (pro)renin receptor controls Wnt signalling: promise from Drosophila and Xenopus.

The (pro)renin receptor (PRR) is a component of the renin-angiotensin system (RAS) that is believed to control blood pressure and salt homeostasis in mammals by favouring tissue activation of RAS. Genetic studies have recently provided novel and exciting insights into how PRR regulates embryonic development in Drosophila and Xenopus through RAS independent functions. By interacting with the H+ vacuolar ATPase (V-ATPase), PRR modulates Wnt signalling pathways. Signalling by Wnt family members governs many aspects of embryonic development and tissue homeostasis. In particular, in mammals, Wnt signalling plays essential roles in the control of stem cell fate decision and lineage commitment in tissues with high self-renewal capacities such as the intestine and the skin, in which we have found PRR to be strongly expressed. Here, we review recent data on how PRR is thought to function during development and place it in the broader context of wnt signalling in skin in general.

Integrins in mammary development.

Integrins are ubiquitously expressed major cell surface receptors for extracellular matrix. Integrin interaction with their extracellular ligands triggers activation of the intracellular signaling pathways that control cell shape, motility, proliferation, survival, cell-type-specific gene expression. In this review, we summarize recent studies analyzing contribution of integrins to the control of the mammary morphogenesis and differentiation, function and maintenance of mammary stem and progenitor cells and resume the data from mouse models revealing the contribution of the integrin-mediated signaling to mammary tumorigenesis.

The mammary myoepithelial cell.

Over the last few years, the discovery of basal-type mammary carcinomas and the association of the regenerative potential of the mammary epithelium with the basal myoepithelial cell population have attracted considerable attention to this second major mammary lineage. However, many questions concerning the role of basal myoepithelial cells in mammary morphogenesis, functional differentiation and disease remain unanswered. Here, we discuss the mechanisms that control the myoepithelial cell differentiation essential for their contractile function, summarize new data concerning the roles played by cell-extracellular matrix (ECM), intercellular and paracrine interactions in the regulation of various aspects of the mammary basal myoepithelial cell functional activity. Finally, we analyze the contribution of the basal myoepithelial cells to the regenerative potential of the mammary epithelium and tumorigenesis.

Control of mammary myoepithelial cell contractile function by α3ß1 integrin signalling.

In the functionally differentiated mammary gland, basal myoepithelial cells contract to eject the milk produced by luminal epithelial cells from the body. We report that conditional deletion of a laminin receptor, α3ß1 integrin, from myoepithelial cells leads to low rates of milk ejection due to a contractility defect but does not interfere with the integrity or functional differentiation of the mammary epithelium. In lactating mammary gland, in the absence of α3ß1, focal adhesion kinase phosphorylation is impaired, the Rho/Rac balance is altered and myosin light-chain (MLC) phosphorylation is sustained. Cultured mammary myoepithelial cells depleted of α3ß1 contract in response to oxytocin, but are unable to maintain the state of post-contractile relaxation. The expression of constitutively active Rac or its effector p21-activated kinase (PAK), or treatment with MLC kinase (MLCK) inhibitor, rescues the relaxation capacity of mutant cells, strongly suggesting that α3ß1-mediated stimulation of the Rac/PAK pathway is required for the inhibition of MLCK activity, permitting completion of the myoepithelial cell contraction/relaxation cycle and successful lactation. This is the first study highlighting the impact of α3ß1 integrin signalling on mammary gland function.

Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited disorder characterized by vascular malformations. Many affected individuals develop recurrent nosebleeds, which can severely affect their quality of life and are clinically difficult to treat. We report here that treatment with thalidomide reduced the severity and frequency of nosebleeds (epistaxis) in the majority of a small group of subjects with HHT tested. The blood hemoglobin levels of the treated individuals rose as a result of reduced hemorrhage and enhanced blood vessel stabilization. In mice heterozygous for a null mutation in the Eng gene (encoding endoglin), an experimental model of HHT, thalidomide treatment stimulated mural cell coverage and thus rescued vessel wall defects. Thalidomide treatment increased platelet-derived growth factor-B (PDGF-B) expression in endothelial cells and stimulated mural cell activation. The effects of thalidomide treatment were partially reversed by pharmacological or genetic interference with PDGF signaling from endothelial cells to pericytes. Biopsies of nasal epithelium from individuals with HHT treated or not with thalidomide showed that similar mechanisms may explain the effects of thalidomide treatment in humans. Our findings demonstrate the ability of thalidomide to induce vessel maturation, which may be useful as a therapeutic strategy for the treatment of vascular malformations.

Expression of the orphan protein Plet-1 during trichilemmal differentiation of anagen hair follicles.

The rat mAb 33A10 recognizes an antigen in a variety of mouse epithelial tissues. In this study, we investigated in detail the expression pattern of the 33A10-defined antigen in the hair follicle. We show that 33A10 reactivity is confined to the most differentiated keratinocytes of the outer root sheath (ORS), the companion layer (CL), and to cells of the sebaceous gland duct. In vitro, the 33A10-defined antigen is expressed in keratinocytes derived from the ORS and accumulates on induction of differentiation. Using microarray analysis and transient transfection approaches, we established that the 33A10-defined antigen is the orphan protein, Placenta-expressed transcript (Plet)-1. Biochemical data indicated that Plet-1 is a glycosylphosphatidylinositol-anchored glycoprotein with N-linked carbohydrates in addition to other posttranslational modifications. Although silencing of Plet-1 expression using stable RNA interference in ORS keratinocytes decreased cellular migration, it increased adhesion to collagens I and IV. Immunohistochemical analysis showed that Plet-1 was primarily localized at the leading edge of epidermal wounds, where keratinocytes contacted the eschar. The restricted localization in both differentiated ORS and CL cells contacting the hair fiber and epidermal wounds suggests a role for the Plet-1 protein in regulating the interaction of keratinocytes with inert tissues.

Adhesion within the stem cell niches.

Growing body of evidence confirms that cell-cell and cell-extracellular matrix adhesion within stem cell niches is essential for the establishment and maintenance of niche architecture, for the generation and transmission of short-distance regulatory signals, and for controlling the frequency and nature of stem cell divisions. Recent studies demonstrated that in many stem cell niches, adhesion to support cells and/or extracellular matrix determines orientation of stem cell division plane, thereby contributing to the control of stem cell self-renewal and differentiation. Thus, although further analysis of the implicated molecular mechanisms is required, cadherin-associated and integrin-associated events appear to play essential regulatory roles in tissue-specific stem cell niches.

Integrin alpha3beta1 inhibits directional migration and wound re-epithelialization in the skin.

Re-epithelialization after skin wounding requires both migration and hyperproliferation of keratinocytes. Laminin-332 is deposited during migration over the provisional matrix. To investigate the function of the laminin-332 binding integrin alpha3beta1 in wound re-epithelialization, we generated Itga3flox/flox; K14-Cre mice lacking the alpha3 subunit specifically in the basal layer of the epidermis. These mice are viable but display several skin defects, including local inflammation, hair loss, basement membrane duplication and microblistering at the dermal-epidermal junction, whereas hemidesmosome assembly and keratinocyte differentiation are not impaired. Wound healing is slightly faster in the absence of integrin alpha3beta1, whereas proliferation, the distribution of other integrins and the deposition of basement membrane proteins in the wound bed are unaltered. In vitro, cell spreading is rescued by increased surface expression of alpha6beta1 integrin in the absence of integrin alpha3. The alpha3-deficient keratinocytes migrate with an increased velocity and persistence, whereas proliferation, growth factor signaling, hemidesmosome assembly, and laminin-332 deposition appeared to be normal. We suggest that integrin alpha3beta1 delays keratinocyte migration during wound re-epithelialization, by binding to the laminin-332 that is newly deposited on the wound bed.

Dual Role of alpha6beta4 integrin in epidermal tumor growth: tumor-suppressive versus tumor-promoting function.

An increased expression of the integrin alpha6beta4 is correlated with a poor prognosis in patients with squamous cell carcinomas. However, little is known about the role of alpha6beta4 in the early stages of tumor development. We have isolated cells from mouse skin (mouse tumor-initiating cells [mTICs]) that are deficient in both p53 and Smad4 and carry conditional alleles of the beta4 gene (Itgb4). The mTICs display many features of multipotent epidermal stem cells and produce well-differentiated tumors after subcutaneous injection into nude mice. Deletion of Itgb4 led to enhanced tumor growth, indicating that alpha6beta4 mediates a tumor-suppressive effect. Reconstitution experiments with beta4-chimeras showed that this effect is not dependent on ligation of alpha6beta4 to laminin-5, but on the recruitment by this integrin of the cytoskeletal linker protein plectin to the plasma membrane. Depletion of plectin, like that of beta4, led to increased tumor growth. In contrast, when mTICs had been further transformed with oncogenic Ras, alpha6beta4 stimulated tumor growth, as previously observed in human squamous neoplasms. Expression of different effector-loop mutants of Ras(V12) suggests that this effect depends on a strong activation of the Erk pathway. Together, these data show that depending on the mutations involved, alpha6beta4 can either mediate an adhesion-independent tumor-suppressive effect or act as a tumor promotor.

Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo.

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5(nlacZ/nlacZ) (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the alpha6beta1 integrin, a laminin receptor, suggesting that integrin alpha6beta1-laminin interactions are required for myotomal laminin matrix assembly. Blocking alpha6beta1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5(nlacZ/nlacZ) embryos. Furthermore, inhibition of alpha6beta1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that alpha6beta1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining alpha6beta1 expression on myogenic precursor cells, and that alpha6beta1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by alpha6beta1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.