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Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy. Functional salivary glands have not been generated from embryonic stem cells (mESCs) to date. Here the authors demonstrate directed in vitro differentiation of mESCs to oral ectoderm and salivary gland rudiments that form mature, functional salivary glands after orthotopic transplantation.

AimTo report the clinical outcomes of autologous minor salivary gland transplantation (MSGT) for the treatment of severe dry eye disease caused by cicatrising conjunctivitis.MethodsThis was a retrospective case series of patients undergoing MSGT at four different centres from 2016 to 2018. The technical modifications included en bloc harvesting of a 20 mm×15 mm mucosa–gland–muscle complex and fixation of the glands to the superior bulbar surface anchored to the superior rectus muscle. The primary outcome measure was improvement in best-corrected visual acuity (BCVA). Secondary outcome measures were change in Schirmer test scores and grades of conjunctival and corneal fluorescein staining, grades of corneal neovascularisation, opacification and keratinisation.Results21 eyes of 19 patients underwent MSGT, with a median follow-up duration of 3 years. The median BCVA improved from a baseline value of 20/500 to 20/125 at 1 year (p=0.0004) and 20/80 at 3 years (p=0.0002) after surgery. The proportion of cases with BCVA ≥20/200 improved from 38% at baseline to 67% at 1 year (p=0.0294), 78% at 2 years (p=0.0227) and 93% at 3 years (p=0.0015) after surgery. There was a significant improvement (p<0.0036) in Schirmer scores, conjunctival and corneal staining scores as well as grades of corneal neovascularisation and opacification after surgery. There were no serious sight-threatening complications in the transplanted eyes or at the donor site.ConclusionsLong-term improvement in the visual acuity, ocular surface environment, and keratopathy was noted after MSGT performed in severely dry eyes using a modified technique.

Salivary gland transfer surgery can reduce xerostomia in oropharyngeal squamous cell carcinoma patients undergoing primary chemoradiation. A potential drawback of salivary gland transfer is the treatment delay associated with the surgery, and its complications. This study aimed to determine whether the treatment delay affects patient survival and to evaluate patient quality of life after salivary gland transfer. A retrospective analysis of 138 patients (salivary gland transfer group, n = 58; non-salivary gland transfer group, n = 80) was performed. Patient survival was compared between these groups using multivariate analysis. Salivary gland transfer patients were further evaluated for surgical complications and for quality of life using the head and neck module of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. Salivary gland transfer and non-salivary gland transfer patients had comparable baseline clinical characteristics. Salivary gland transfer patients experienced a median treatment delay of 16.5 days before chemoradiation (p = 0.035). Multivariate analysis showed that this did not, however, correspond to a survival disadvantage (p = 0.24 and p = 0.97 for disease-free and disease-specific survival, respectively). A very low complication rate was reported for the salivary gland transfer group (1.7 per cent). Questionnaire scores for the item 'xerostomia' were very low in salivary gland transfer patients. The treatment delay associated with salivary gland transfer surgery does not negatively affect patient survival. Oropharyngeal squamous cell patients have an excellent quality of life after salivary gland transfer.

Liver disease is a serious problem affecting millions of people with continually increasing prevalence. Stem cell therapy has become a promising treatment for liver dysfunction. We previously reported on human minor salivary gland mesenchymal stem cells (hMSGMSCs), which are highly self-renewable with multi-potent differentiation capability. In this study, keratinocyte-like cells with self-regeneration and hepatic differentiation potential were isolated and characterized, and named human minor salivary gland epithelial progenitor cells (hMSG-EpiPCs). hMSG-EpiPCs were easily obtained via minor intraoral incision; they expressed epithelial progenitor/stem cell and other tissue stem cell markers such as CD29, CD49f, cytokeratins, ABCG2, PLET-1, salivary epithelial cell markers CD44 and CD166, and the Wnt target related gene LGR5 and LGR6. The cells were induced into functional hepatocytes in vitro which expressed liver-associated markers ALB, CYP3A4, AAT, and CK18. Upon transplantation in vivo , they ameliorated severe acute liver damage in SCID mice caused by carbon tetrachloride (CCl 4 ) injection. In a two-thirds partial hepatectomy mouse model, the transplanted cells survived at least 4 weeks and exhibited hepatic potential. These findings demonstrate that hMSG-EpiPCs have potential as a cellular therapy basis for hepatic diseases, physiological and toxicology studies and regenerative medicine.

Salivary glands (SGs) secrete more than half a liter of saliva daily. Saliva has many functions in maintaining the normal homeostasis of the oral cavity. Several causes underlie salivary impairment, where irradiation therapy to head and neck cancer patients is one of the most debilitating causes leading to considerable decrease in the patients' quality of life. In the last decade, others and we have focused on implementing tissue engineering principles combined with gene transfer and stem cell methodologies to develop an artificial SG device. This manuscript provides an overview of the current status of engineering an artificial SG.

ObjectiveTo evaluate minor salivary glands and labial mucous membrane graft in patients with severe symblepharon and dry eye secondary to Stevens–Johnson syndrome (SJS).MethodsA prospective, non-comparative, interventional case series of 19 patients with severe symblepharon and dry eye secondary to SJS who underwent labial mucous membrane and minor salivary glands transplantation. A complete ophthalmic examination including the Schirmer I test was performed prior to and following surgery. All patients had a preoperative Schirmer I test value of zero.ResultsNineteen patients with severe symblepharon and dry eye secondary to SJS were included in the study. There was a statistically significant improvement in the best spectacle-corrected visual acuity in eight patients (t test; p=0.0070). Values obtained in the Schirmer I test improved significantly in 14 eyes (73.7%) 6 months following surgery (χ2 test; p=0.0094). A statistically significant increase in tear production (Schirmer I test) was found in eyes that received more than 10 glands per graft compared with eyes that received fewer glands (χ2 test; p=0.0096). Corneal transparency improved significantly in 11 (72.2%) eyes and corneal neovascularisation improved significantly in five eyes (29.4%) (McNemar test; p=0.001 and p=0.0005). The symptoms questionnaire revealed improvement in foreign body sensation in 53.6% of the patients, in photophobia in 50.2% and in pain in 54.8% (Kruskal–Wallis test; p=0.0167).ConclusionLabial mucous membrane and minor salivary glands transplantation were found to constitute a good option for the treatment of severe symblepharon and dry eye secondary to SJS. This may be considered as a step prior to limbal stem cell and corneal transplantation in these patients.

The hypothesis that replication-competent mouse cytomegalovirus (MCMV) is detectable in severe combined immunodeficient (scid) mice after implantation of latently infected tissue was examined. Sections of latently infected salivary gland from 5 BALB/c mice were implanted into 20 C.B-17 scid mice. Recipient scid mice were sacrificed weekly for 5 weeks, and MCMV infection was detected in target organs using culture and DNA polymerase chain reaction (PCR). All donors were negative by standard culture but positive by DNA PCR. Replicating MCMV was recovered from 9 of 15 recipient scid mouse salivary gland, lung, liver, or spleen samples at postoperative weeks 2–5. No virus was recovered from recipient scid mice at weeks 1 or 12. Transplantation of latently infected tissues into scid mice resulted in rapid reactivation and dissemination of the virus. Further study of this model promises insight into the mechanisms of CMV latency and reactivation.

An essential structural feature of fluid-secreting epithelial tissues is the presence of tight junctions. To develop a tissue-engineered organ capable of fluid secretion, the cellular component must establish these structures. As part of efforts to create an engineered artificial salivary gland, we have examined the ability of a candidate allogeneic graft cell line, HSG, to produce several key tight junction proteins, as well as to exhibit functional activities consistent with effective tight junction strand formation. In contrast to results obtained with a control kidney cell line, MDCK-II, HSG cells were unable to synthesize four important tight junction-associated proteins: ZO-1, occludin, claudin-1, and claudin-2. In addition, unlike MDCK-II cells, HSG cell monolayers could not restrict paracellular permeability. HSG cells were, thus, unable to generate significant transepithelial electrical resistance or serve as an effective barrier to osmotically imposed fluid movement. Furthermore, these two functional activities could not be reconstituted via the stable transfection of HSG cells with cDNAs encoding either claudin-1 or claudin-2. We conclude that because of their inability to form tight junctions, HSG cells are unsuitable for use as an allogeneic graft cell in an artificial salivary fluid secretory device. These studies also emphasize the importance of graft cell selection in artificial organ development, as certain required characteristics may be difficult to reengineer.

The purpose of this study was to examine the growth and key functional abilities of primary cultures of salivary epithelial cells toward developing an artificial salivary gland. Cultures of epithelial cells originating from submandibular glands of BALB/c mice were established. Parenchymal cells were isolated by a Percoll gradient technique and thereafter seeded on irradiated NIH 3T3 fibroblasts serving as a feeder layer. The isolated cells were termed autologous salivary gland epithelial (ASGE) cells and could be cultivated for at least five passages (time limit of experiments). ASGE cells presented the typical organizational behavior of epithelial cells and electron microscopy, as well as immunostaining for cytokeratins, confirmed their epithelial origin. Furthermore, measurements of transepithelial resistance and water permeability indicated the ability of the ASGE cells to form a functional epithelial barrier. This study suggests that primary salivary epithelial cells can be obtained that exhibit critical characteristics needed for use with an artificial secretory device.

Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland). We are using the well-studied HSG salivary cell line as a potential allogeneic graft cell for this device. One drawback of using a cell line is the potential for malignant transformation. If such an untoward response occurred, the device could be removed. However, in the event that any HSG cells escaped, we wished to provide additional patient protection. Accordingly, we have engineered HSG cells with a hybrid adeno-retroviral vector, AdLTR.CMV-tk, to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene as a novel safety factor. Cells were grown on plastic plates or on poly-L-lactic acid disks and then transduced with different multiplicities of infection (MOIs) of the hybrid vector. Thereafter, various concentrations of ganciclovir (GCV) were added, and cell viability was tested. Transduced HSG cells expressed HSV-tk and were sensitive to GCV treatment. Maximal effects were seen at a MOI of 10 with 50 μM of GCV, achieving 95% cell killing on the poly-L-lactic acid substrate. These results suggest that engineering the expression of a suicide gene in an allogeneic graft cell may provide additional safety for use in an artificial salivary gland device.