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Generation of Airway Epithelial Cell Air-Liquid Interface Cultures from Human Pluripotent Stem Cells

Diseases of the conducting airway such as asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and viral respiratory infections are major ...

Boston University School of Medicine

Boston University and Boston Medical Center

Pulmonology in Pregnancy.

Mutations in the cystic fibrosis transmembrane conductance regulator () gene cause the life-limiting hereditary disease, cystic fibrosis (CF). Decreased or absent functional CFTR protein in airway epithelial cells leads to abnormally viscous mucus and impaired mucociliary transport, resulting in bacterial infections and inflammation causing progressive lung damage. There are more than 2000 known variants in the  gene. A subset of CF individuals with specific  mutations qualify for pharmacotherapies of variable efficacy. These drugs, termed CFTR modulators, address key defects in protein folding, trafficking, abundance, and function at the apical cell membrane resulting from specific  mutations. However, some  mutations result in little or no CFTR mRNA or protein expression for which a pharmaceutical strategy is more challenging and remote. One approach to rescue CFTR function in the airway epithelium is to replace cells that carry a mutant  sequence with cells that express a normal copy of the gene. Cell-based therapy theoretically has the potential to serve as a one-time cure for CF lung disease regardless of the causative  mutation. In this review, we explore major challenges and recent progress toward this ambitious goal. The ideal therapeutic cell would: (1) be autologous to avoid the complications of rejection and immune-suppression; (2) be safely modified to express functional CFTR; (3) be expandable  to generate sufficient cell quantities to restore CFTR function; and (4) have the capacity to engraft, proliferate and persist long-term in recipient airways without complications. Herein, we explore human bronchial epithelial cells (HBECs) and induced pluripotent stem cells (iPSCs) as candidate cell therapies for CF and explore the challenges facing their delivery to the human airway.

Challenges Facing Airway Epithelial Cell-Based Therapy for Cystic Fibrosis.

Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2 iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.

Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells.

The unprecedented public health burdens of coronavirus disease (COVID-19) have intensified the urgency of identifying effective, low-cost treatments that limit the need for advanced life support measures and improve clinical outcomes. However, personal protective equipment and staffing shortages, disease virulence, and infectivity have created significant barriers to traditional clinical trial practices. We present the novel design of a pragmatic, adaptive, multicenter, international, prospective randomized controlled clinical trial evaluating the safety and effectiveness of awake prone positioning in spontaneously breathing patients with COVID-19 (APPEX-19 [Awake Prone Position for Early Hypoxemia in COVID-19]). Key innovations of this trial include ) a novel smartphone-based communication process that facilitates rapid enrollment and intervention delivery while allowing social distancing and conservation of personal protective equipment, ) Bayesian response-adaptive randomization to allow preferential assignment to the most effective intervention and expedite trial completion compared with frequentist designs, ) remote electronic collection of patient-reported outcomes and electronic medical record data, and ) pragmatic prospective use of patient-reported data and data collected as part of routine clinical care. Clinical trial registered with www.clinicaltrials.gov (NCT04344587).

Article	Year
Pulmonology in Pregnancy. American journal of respiratory and critical care medicine\| PubMed ID: 29155600	2018
Challenges Facing Airway Epithelial Cell-Based Therapy for Cystic Fibrosis. Frontiers in pharmacology\| PubMed ID: 30800069	2019
Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells. Nature communications\| PubMed ID: 31924806	2020
Rationale and Design of the Awake Prone Position for Early Hypoxemia in COVID-19 Study Protocol: A Clinical Trial. Annals of the American Thoracic Society\| PubMed ID: 33647225	2021

Andrew Berical

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