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Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1+ lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.

Although Hertwig’s epithelial root sheath (HERS) performs an important function in the formation of the tooth root, the developmental mechanisms that control HERS growth and differentiation remain to be thoroughly elucidated. Bone morphogenetic protein 4 (BMP4), which is secreted by mesenchymal cells, acts on the dental epithelium as a regulator of cell differentiation during crown formation. In an effort to determine whether BMP4 specifically regulates the development of HERS in the dental epithelium, we assessed the localizations of BMP4, BMP receptor-IB (BMPR-IB), and BMPR-II during molar root formation in the mouse. HERS cells were shown to express BMPR-IB and BMPR-II. BMP4-positive cells were detected densely in the dental papillae around HERS, thereby suggesting that BMP4 participated in HERS formation. Beads soaked in BMP4, NOGGIN, or phosphate-buffered saline (PBS) were implanted into the pulp cavity under culture conditions, and the length of HERS was evaluated with regard to the proliferating cells. After 12 h, both groups exhibited a similar HERS developmental pattern, with the length and shape of HERS bearing a close resemblance to one another. However, after 48 h, the observed HERS elongation was significantly shorter in the BMP4-treated group. In addition, proliferative cell nuclear antigens were detectable only in the NOGGIN- and PBS-treated groups. These findings demonstrate that mesenchymally expressed BMP4 regulates HERS development by preventing elongation and maintaining cell proliferation. BMP4 may, therefore, prove useful as a root-formation regulatory agent in a variety of tissue-engineering applications.

Recent studies have focused on understand lung repair mechanisms, which are critical for treating respiratory diseases. In this study, we develop alveolar organoids to investigate the complex interactions between alveolar epithelial cells, niche fibroblasts and macrophages, which are essential for lung development, maintenance and repair, especially under physiological injury. Our results suggest that alveolar organoids may be a model for epithelial cell regeneration and the inflammatory response in lung tissue. Alveolar organoid studies can also serve as models for various lung injuries and demonstrate mechanisms in the injured human lung.

Human retinal pigment epithelial (RPE) cells produce levodopa and their transplantation into the striatum might improve continuity of administration compared with that achieved with oral levodopa. We aimed to assess the safety, tolerability, and efficacy of transplantation of microcarrier-bound human RPE cells versus a sham surgery control in patients with advanced Parkinson's disease. In this randomised, double-blind study eligible patients were aged 36–70 years, had been symptomatic for at least 5 years, were in Hoehn and Yahr stage 3–4 and had unified Parkinson's disease rating scale (UPDRS) motor scores of 38–70 when off medication (off state), and had symptoms that responded to oral levodopa but were insufficiently controlled by optimised pharmacotherapy. Randomisation was done in a 1:1 ratio. Only the neurosurgical team was aware of treatment assignments. During stereotactic transplantation around 325 000 cells per side were injected into the postcommissural putamen; sham surgery patients received partial burr holes. The primary efficacy endpoint was change in UPDRS off-state motor score at 12 months. This study is registered with ClinicalTrials.gov, number NCT00206687. Of 71 enrolled patients, 35 underwent cell transplantation and 36 sham surgery. Change in mean motor scores did not differ significantly between groups (−10·5 [SD 10·26] for transplantation vs −10·1 [SD 12·26] for sham surgery, p=0·9). The overall rate of adverse events was similar in the two study groups, although the number attributable to surgery or RPE cells (mostly neurological or psychiatric) was higher in transplant recipients. Two and seven patients died in the sham surgery and transplantation group, respectively; one death in the latter group was possibly related to surgery or RPE cells. Transplantation of human RPE cells provided no antiparkinsonian benefits compared with sham surgery. Bayer HealthCare AG.

Background To determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency. Methods In vitro cellular outgrowth and phenotype of the limbal epithelial cell and composite grafts were validated using a new protocol. Patients received either autologous (n = 15) or allogenic (n = 3) explants cultured using a standardized protocol free from xenogenic products. The resulting grafts were transplanted using a reduced manipulation surgical technique. Results The majority of cells (>50%) displayed a progenitor phenotype typified by positive immunofluorescence for ∆Np63, CK14 and ABCG2 and low immunofluorescence for CK3/12 and desmoglein 3 proteins. The surgical protocol was designed to minimize manipulation and the graft itself was secured without sutures. The transplant recipients were followed for a mean of 24 months. Twelve of the 18 transplant recipients were graded as anatomically successful (67%), based on the defined success parameters. There was a significant reduction in corneal neovascularization, which was accompanied by an improvement in pain though not photophobia or central corneal opacity post transplant. The transplantation protocol showed no measureable effect on visual acuity. Conclusion We conclude that this standardized culture system and surgical approach is safe and effective in reducing corneal neovascularization. The technique is free from animal contaminants and maintains a large proportion of progenitor cells. Although this technique did not improve visual function, restoring a functional epithelial cell layer and reducing corneal neovascularization provides an improved platform for a penetrating keratoplasty to ultimately improve visual function.

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation. RPAP3 is a subunit of the R2TP complex, a co-chaperone of HSP90, with substrate proteins involved in transcription, ribosome biogenesis, DNA repair and cell growth. Here the authors report that deletion of Rpap3 abrogates cell proliferation and homeostasis in mouse intestine, partly through destabilization of PI3K-like kinases, while elevated RPAP3 levels in colorectal tumors are associated with poor prognosis.

Alveolar epithelial cells are critical to the pathogenesis of pulmonary inflammation and fibrosis, which are associated with overexpression of type 2 cytokine IL-13. IL-13 is known to induce the production of profibrotic (e.g., periostin) and pro-inflammatory (e.g., eotaxin-3) mediators in human airway epithelial cells, but it remains unclear if human primary alveolar epithelial cells increase periostin and eotaxin expression following IL-13 stimulation. The goals of this study are to determine if alveolar epithelial cells increase periostin and eotaxin expression upon IL-13 stimulation, and if alveolar and airway epithelial cells from the same subjects have similar responses to IL-13. Paired alveolar and airway epithelial cells were isolated from donors without any lung disease, and cultured under submerged or air-liquid interface conditions with or without IL-13. Up-regulation of periostin protein and mRNA was observed in IL-13-stimulated alveolar epithelial cells, which was comparable to that in IL-13-stimulated paired airway epithelial cells. IL-13 also increased eotaxin-3 expression in alveolar epithelial cells, but the level of eotaxin mRNA was lower in alveolar epithelial cells than in airway epithelial cells. Our findings demonstrate that human alveolar epithelial cells are able to produce periostin and eotaxin in responses to IL-13 stimulation. This study suggests the need to further determine the contribution of alveolar epithelial cell-derived mediators to pulmonary fibrosis.

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of 'client' proteins. How ubiquitous chaperone machineries direct their activities towards specific sets of substrates is unclear. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone that confers specificity for the FKBP8-HSP90 chaperone complex towards axonemal dynein clients required for cilia motility. Loss of ZMYND10 perturbs the chaperoning of axonemal dynein heavy chains, triggering broader degradation of dynein motor subunits. We show that pharmacological inhibition of FKBP8 phenocopies dynein motor instability associated with the loss of ZMYND10 in airway cells and that human disease-causing variants of ZMYND10 disrupt its ability to act as an FKBP8-HSP90 co-chaperone. Our study indicates that primary ciliary dyskinesia (PCD), caused by mutations in dynein assembly factors disrupting cytoplasmic pre-assembly of axonemal dynein motors, should be considered a cell-type specific protein-misfolding disease.

Identification of appropriate donor cell types is important for lung cell therapy and for lung regeneration. Previous studies have indicated that mesenchymal stromal cells derived from human bone marrow (hBM-MSCs) and from human adipose tissue (hAT-MSCs) may have the ability to trans-differentiate into lung epithelial cells. However, these data remain controversial. Herein, the ability of hBM-MSCs and hAT-MSCs to repopulate acellular rodent lung tissue was evaluated. hBM-MSCs and hAT-MSCs were isolated from bone marrow aspirate and lipoaspirate, respectively. Rat lungs were decellularized with CHAPS detergent, followed by seeding the matrix with hBM-MSCs and hAT-MSCs. Under appropriate culture conditions, both human MSC populations attached to and proliferated within the lung tissue scaffold. In addition, cells were capable of type 2 pneumocyte differentiation, as assessed by marker expression of surfactant protein C (pro-SPC) at the protein and the RNA level, and by the presence of lamellar bodies by transmission electron microscopy. Additionally, hAT-MSCs contributed to Clara-like cells that lined the airways in the lung scaffolds, whereas the hBM-MSCs did not. We also tested the differentiation potential of MSCs on different extracellular matrix components in vitro , and found that protein substrate influences MSC epithelial differentiation. Together our data show the capacity for human MSCs to differentiate toward lung epithelial phenotypes, and the possibility of using these cells for lung cell therapies and tissue engineering.

Limbal stem cell deficiency (LSCD) is a disease resulting from the loss or dysfunction of epithelial stem cells, which seriously impairs sight. Autologous limbal stem cell transplantation is effective in unilateral or partial bilateral disease but not applicable in total bilateral disease. An allogeneic source of transplantable cells for use in total bilateral disease can be obtained from culture of donated cadaveric corneal tissue. We performed a controlled multicenter study to examine the feasibility, safety, and efficacy of allogeneic corneal epithelial stem cells in the treatment of bilateral LSCD. Patients were randomized to receive corneal epithelial stem cells cultured on amniotic membrane (AM): investigational medicinal product (IMP) or control AM only. Patients received systemic immunosuppression. Primary endpoints were safety and visual acuity, secondary endpoint was change in composite ocular surface score (OSS). Sixteen patients were treated and 13 patients completed all assessments. Safety was demonstrated and 9/13 patients had improved visual acuity scores at the end of the trial, with no significant differences between IMP and control groups. Patients in the IMP arm demonstrated significant, sustained improvement in OSS, whereas those in the control arm did not. Serum cytokine levels were measured during and after the period of immune suppression and we identified strongly elevated levels of CXCL8 in the serum of patients with aniridia, which persisted throughout the trial. This first randomized control trial of allogeneic corneal epithelial stem cells in severe bilateral LSCD demonstrates the feasibility and safety of this approach. Stem Cells Translational Medicine 2019;8:323–331 Patients with severe ocular surface disorder received transplants of amniotic membrane with (black bars) or without (gray bars) cadaveric‐donor‐derived cultured limbal stem cells. All patients received immune suppression. Only patients who received transplants containing limbal stem cells showed sustained significant improvements (reductions) in combined ocular surface scores (5 factors scored 0–3 where 0 is a normal eye score).