Laboratory for Oral Molecular Biology

Craniofacial development represents one of the most intricate developmental processes of our body. It involves numerous morphogenetic movements encompassing each of the three germ layers and several spatiotemporally-regulated signaling pathways. This complexity makes the morphogenesis of the craniofacial structures highly susceptible to dysregulations.

Cleft lip/palate (CLP) is a common anomaly affecting the upper lip and/or the palate. The complexity and frequency of this craniofacial malformation together with the intensive and multidisciplinary treatment approach, starting from birth until adulthood, make CLP a major psychological, social, and economic burden for affected individuals, their parents, and society as a whole.

Additionally, while the surgical techniques to correct clefts have been optimized, clefting is still associated with increased infant mortality, significant morbidity and with multiple complications, such as the formation of excessive scar tissue, which can lead to the impaired mid-face growth after palate repair.

We take a patient-oriented and collaborative research approach to anticipate a better understanding of the genetic and molecular mechanisms involved in the pathogenesis of CLP, with the goal to help future CLP-affected children.

Research interests

The lack of clinically relevant human study tools to better understand the genes and their functions involved in the pathogenesis of CLP prompted us to establish a cell bank of CLP patient-derived fibroblasts and keratinocytes. During standard surgical closure of the lip at the age of 3-5 months, the marginal portion of the upper lip covering the cleft is in excess and needs to be removed. With the approval of the Kantonale Ethikkommission of Bern, Switzerland (protocol number: 2017-03194), we started to collect such discarded cleft lip tissues to isolate primary cells.

We developed a very robust and reproducible explant culture assay for the isolation of tissue-derived cells. We take every effort possible to reduce any inter-experimental variations during primary cell isolation: tissues are processed within less than 1 h after biopsy, explant cultures are performed by two lab members only, and our internal protocol is strictly followed. Prior to freezing, all primary cells are tested for mycoplasma contamination by a PCR-based mycoplasma detection assay (Praetorius, 2015) and by DAPI staining of cultures that had been grown in the absence of any antibiotics for at least 5 days.

At the moment our cell bank consists of about 50 CLP patient-derived cells, in addition to a collection of healthy control cells, mainly from the orofacial region, but also from other anatomical parts.

With our cell bank we aim to

  • gain a better understanding of the complex molecular processes underlying CLP, which might help future CLP patients
  • foster collaborations and provide ethically obtained and clinically relevant cell systems to the research community
  • establish a personalized cell depository, which might be beneficial for future translational applications 

This project is in collaboration with the Pediatric Surgery Department (Dr. med. I. Schnyder), University Hospital Bern, Bern, Switzerland

Van der Woude syndrome (VWS, OMIM #119300) is the most common cleft syndrome accounting for 2% of all cleft cases with a prevalance of 1/35'000 to 1/100'000 newborns. Many VWS individuals (30-79%) present with very characteristic depression (pits) near the center of the lower lip in combination with CLP or cleft palate only. The condition is caused by gene mutations and is inherited in an autosomal dominant way.

Initially, the VWS locus was mapped to human chromosome 1q32-q41 and the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) was found to be mutated in around 72% of VWS cases (Kondo et al., 2002). Later, dominant-negative mutations within the transcription factor grainyhead like transcription factor 3 (GRHL3), a direct target gene of IRF6, have been identified in VWS-affected families not having pathogenic mutations in IRF6 (Peyrard-Janvid et al., 2014). More recently, a third VWS-associated gene has been discovered: a rare missense mutation within NME1, encoding for an IRF6-interacting proteins, was found to cause VWS in individuals not having IRF6 and GRHL3 mutations (Parada-Sanchez et al., 2017).

Most literature is available on the function of IRF6 in VWS: Keratinocytes isolated from skin biopsies from the hip region of VWS individuals (harboring IRF6 mutations) exhibit an increased proliferation rate (Hixon et al., 2016) and VWS patients experience more wound healing complications following repair of their clefts (Jones et al., 2010) compared to non-syndromic CLP keratinocytes and patients, respectively. Up to now, more than 200 mutations within IRF6 have been found associated with either VWS, popliteal pterygium syndrome (PPS, OMIM #119500), which can mimic the allelic disorder VWS in mild cases, or with non-syndromic CLP. However, the underlying mechanistic link between gene mutation and phenotype remains elusive for most of them.

Using a combination of genetics, molecular and cellular biology we aim to establish genotype-phenotype correlations in VWS-patients, which should help to understand gene function in health and disease.

In collaboration with: PD. A. Schaller, Division of Human Genetics, Children's Hospital Bern, Switzerland and I. Schnyder, Pediatric Surgery Department, Children's Hospital Bern, Switzerland

Although a number of genes have been linked to cleft lip/palate (CLP), which is one of the most common congenital craniofacial malformations, the genetic cause is only known for a minority of the cases. A fraction of CLP patients undergoing surgical repair of the cleft suffers from excessive scar formation, which later can interfere with maxillary growth. The impaired wound healing procedure may be attributed to the differential gene expression of those patients who are prone to develop excessive scarring.

  • To elucidate the contribution of the genetic background to the abnormal healing profile in vitro of CLP patients, a genotype-phenotype correlation of CLP patient-derived cells is attempted. The expression profiles of genes that are related to wound healing, extracellular matrix formation, and cleft lip/palate are being assessed in order to unravel a possible link between the aforementioned processes. Patient-derived cells can be categorized into different subgroups, based on their differential gene expression correlated to their behavior and motility.
  • Since wound healing complications are observed in 30% of CLP patients undergoing primary cleft surgery, we hypothesize that certain CLP-associated transcription factors might be responsible for the impaired wound healing. We focus on the transcription factors IRF6 and GRHL3 that have been found mutated in CLP, are involved in wound healing and are also supposed to act as tumor suppressors. Since tumors are similar to non-healing wounds and misuse developmental signalling pathways for their manifestation, we speculate that mutations in these genes might represent common factors underlying the development of CLP, wound healing complications and cancer. The two genes of interest are knocked out with CRISPR-Cas9 in keratinocyte cell lines in order to elucidate their function in regard to proliferation, differentiation and migration potential.

In CLP patients, alveolar cleft, or the discontinuity of the maxillary arch, is one of the most common clinical manifestations. In these cases, grafting is an essential procedure in order to reestablish the integrity of the alveolar bone allowing proper tooth eruption.

During the closure of the cleft lip, which is performed at the age of 3-5 months, lip tissue fragments have to be excised by the surgeon in order to achieve proper lip reconstruction. These discarded fragments might represent an optimal source of cells with regenerative potential. As an alternative to bone grafts, we aim to use these autogenous cells as a new source for the regeneration of new bone and other tissues in CLP patients.

Our translational approach offers the possibility to develop a personalized therapeutic strategy for CLP patients with the potential to lower their burden of care by reducing the number of required invasive surgeries.

Epithelia are organized as tightly packed cell sheets that line internal cavities and cover most of our body and organ surfaces. According to the shape of the cells, epithelial tissues can be classified into squamous, cuboidal or columnar single- (simple) or multi-layered (stratified) epithelium.

Our largest organ is the skin, which serves various functions, including thermoregulation and protection from the environment. The skin consists of three layers: 1) epidermis, 2) dermis, and 3) hypodermis. The epidermis is the outermost layer and is mainly composed of keratinocytes that form a stratified and keratinized tissue. In contrast, the oral cavity is mostly composed of stratifying, but non-keratinizing keratinocytes.

Historically, animal models have been used to study epithelial biology or to test toxicity and efficacy of drugs and/or cosmetics. However, ethical concerns prompted the search for novel, animal-free models that mimic the in vivo skin/oral mucosa as much as possible. 3D organotypic skin/mucosa cultures appear to be appealing models as they closely mimic the in vivo structure of the respective epithelium.

Our approach includes the use of primary patient-derived cells for the establishment of 3D models. We isolate primary keratinocytes and fibroblasts from the same donor and create a living cell repository of the cranio-/orofacial region (Parisi et al., 2021). Using such cells, we aim to establish 3D skin/oral mucosa and full-thickness skin/oral mucosa models and characterize them for their usefulness as alternatives to animal in vivo models. With this approach, we intend to prevent unnecessary animal experimentations. In addition, we make use of these models to elucidate the roles of specific CLP candidate genes (e.g., IRF6 and GRHL3) in regulating epithelial tissue homeostasis.

Briefly, we aim to:

  • Establish 3D cell models depicting the various tissue types of the cranio-/orofacial region (oral mucosa, gingiva, lip, skin).
  • Study the communication and interaction between fibroblasts and keratinocytes.
  • Assess the importance of the fibroblasts to provide information for epithelial identity.
  • Provide personalized CLP models to study specific gene functions and CLP-related complications, such as altered wound healing.
N/TERT1 H&E staining
N/TERT1 Immunohistochemistry for Ki67
N/TERT1 Immunohistochemistry for Involucrin
N/TERT1 Immunohistochemistry for Loricrin