Stem cells, organoids, other cell subjects

There were more than 150 references to “stem cells and cystic fibrosis” by 2013. As their use in treating people with CF some way off only a few are mentioned here.

2005 Wang G, Bunnell BA, Painter RG, Quiniones BC, Tom S, Lanson NA Jr, Spees JL Bertucci D, Peister A, Weiss DJ, Valentine VG, Prockop DJ Kolls JK. Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis. Proc Nat Acad Sci 2005; 102:186- 191. [PubMed]
The authors explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. They found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivococulture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.

So stem cells from the marrow of people with CF could be treated with gene therapy and still develop into airway epithelia and function as chloride channels when they reached the lungs. Another attractive potential method of correcting the basic defect in respiratory epithelium.

2005 Spencer H, Rampling D, Aurora P, Bonnet D, Hart SL, Jaffe A. Transbronchial biopsies provide longitudinal evidence for epithelial chimerism in children following sex mismatched lung transplantation. Thorax 2005; 60:60-62. [PubMed]
The aim of this study was to assess whether transbronchial biopsies could be used to study the time course of chimerism following lung transplantation (chimerism = the presence in an individual cells of different origins). Specimens of transbronchial lung biopsies from five time points taken for clinical purposes from two boys, who had received a sex mismatched heart-lung transplant for end stage CF, were examined. Evidence of chimerism was found in up to 6.6% of epithelial cells in bronchial tissue without apparent evidence of fusion. This engraftment was seen as early as 3 weeks and remained relatively constant up to 37 months.

This study has demonstrated proof of principle for long term chimerism in lung epithelium. Transbronchial biopsies may provide a new method for studying the kinetics of stem cell engraftment in the lung.

2005 Coraux C. Nawrocki-Raby B. Hinnrasky J. Kileztky C. Gaillard D. Dani C. Puchelle E. Embryonic stem cells generate airway epithelial tissue. Am J Resp Cell Mol Biol 2005; 32:87-92. [PubMed]
Embryonic stem (ES) cells are self-renewable and pluripotent cells derived from the inner cell mass of a blastocyst-stage embryo. ES cell pluripotency is being investigated increasingly to obtain specific cell lineages for therapeutic treatments and tissue engineering. Type II alveolar epithelial cells have been derived from murine ES cells, but the capacity of the latter to generate differentiated airway epithelial tissue has never been reported. Herein, we show by RT-PCR and immunocytochemistry that murine ES cells are able to differentiate into nonciliated secretory Clara cells, and that type I collagen induces this commitment. Moreover, when cultured at the air-liquid interface, ES cells give rise to a fully differentiated airway epithelium. By quantitative histologic examination, immunohistochemistry, and scanning electron microscopy, we show that the bioengineered epithelium is composed of basal, ciliated, intermediate, and Clara cells, similar to those of native tracheobronchial airway epithelium. Transmission electron microscopy and Western blotting reveal that the generated epithelium also exhibits the ultrastructural features and secretory functions characteristic of airway epithelial tissue. These results open new perspectives for cell therapy of injured epithelium in airway diseases, such as bronchopulmonary dysplasia, cystic fibrosis, or bronchiolitis obliterans.

2005 Wang G. Bunnell BA. Painter RG. Quiniones BC. Tom S. Lanson NA Jr. Spees JL. Bertucci D. Peister A. Weiss DJ. Valentine VG. Prockop DJ. Kolls JK. Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: potential therapy for cystic fibrosis. Proc Nat Acad Sci 2005; 102:186-191. [PubMed] Marrow stromal stem cells (MSCs) possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl(-) secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.

2008 Sueblinvong V, Loi R, Eisenhauer PL, Bernstein IM, Suratt BT, Spees JL, Weiss DJ. Derivation of lung epithelium from human cord blood-derived mesenchymal stem cells. Am J Resp Crit Care 2008; 177:701-711. [PubMed]
Both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and non-hematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases.
Human cord blood was obtained from normal deliveries at the University of Vermont. Cord blood-derived mesenchymal stem cells (MSCs) were cultured in specialized airway growth media or with specific growth factors. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. The MSCs were systematically administered to immunotolerant, non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice and their lungs were analyzed for the presence of human cells. When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed a variety of protein including CFTR. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression. The authors concluded that cord blood stem cells appear to be comparable to marrow-derived stem cells in their ability to express phenotypic markers of airway epithelium and to participate in airway remodelling in vivo.

There is no way to describe this study briefly and it appears to be potentially important providing as it does further evidence that stem cell therapy may be a possibility for cystic fibrosis. Cord blood-derived mesenchymal stem cells appear to be comparable to mesenchymal stem cells obtained from adult bone marrow in their ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo. This is relevant particularly for those CF families that have already arranged to store umbilical cord blood from any subsequent pregnancies in the hope that it may prove to be a source of stem cells which could be used to treat their previous child with CF.

2012 Wong AP, Bear CE, Chin S, Pasceri P, Thompson TO, Huan LJ, Ratjen F, Ellis J, Rossant J.Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol. 2012; 30(9):876-82. [PubMed]
The authors describe an in vitro directed differentiation protocol for generating functional CFTR-expressing airway epithelia from human embryonic stem cells. Carefully timed treatment by exogenous growth factors that mimic endoderm developmental pathways in vivo followed by air-liquid interface culture results in maturation of patches of tight junction–coupled differentiated airway epithelial cells that demonstrate active CFTR transport function. As a proof of concept, treatment of CF patient induced pluripotent stem cell–derived epithelial cells with a small-molecule compound to correct for the common CF processing mutation resulted in enhanced plasma membrane localization of mature CFTR protein.
The study provides a much needed method for generating patient-specific airway epithelial cells for disease modeling and in vitro drug testing.

2014 Firth AL. Dargitz CT. Qualls SJ. Menon T. Wright R. Singer O. Gage FH. Khanna A. Verma IM. Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells. Proc Natl Acad Sci USA 2014; 111(17):E1723-30. (free full text) [PubMed]
Using induced pluripotent stem cells (iPSCs), the authors generated mature multi-ciliated cells in a functional airway epithelium.
Their report demonstrating the generation of mature multi-ciliated cells in respiratory epithelium from iPSCs is a significant advance toward modelling a number of human respiratory diseases in vitro.

Boruczkowski D; Gladysz D; Demkow U; Pawelec K.  The Potential of Wharton’s Jelly Derived Mesenchymal Stem Cells in Treating Patients with Cystic Fibrosis. Adv Exp Med Biol 2015; 833:23-9.  [PubMed]
Even though CF is monogenic, the trials of topical gene transfer into airway epithelial cells have so far been disappointing. It is proven that stem cells can be differentiated into type II alveolar epithelial cells. Wharton’s jelly-derived mesenchymal stem cells (MSC) from non-CF carrier third-party donors could be an effective alternative to bone marrow or embryonic stem cells.  This review discusses the clinical potential of mesenchymal stem cell in CF patients.

Danya Muilwijk, Eyleen de Poel, Peter van Mourik, Sylvia W F Suen, Annelotte M Vonk , Jesse E Brunsveld  et al.(please see PubMed abstract for full author list).  Forskolin-induced Organoid Swelling is Associated with Long-term CF Disease Progression.  Eur Respir J 2022 Jan 27;2100508.doi: 10.1183/13993003.00508-2021. Online ahead of print.  3  [Pubmed]

Rationale: Cystic fibrosis (CF) is a monogenic life-shortening disease associated with highly variable individual disease progression which is difficult to predict. Here we assessed the association of forskolin-induced swelling (FIS) of patient-derived organoids (PDO) with long-term CF disease progression in multiple organs and compared FIS with the golden standard biomarker sweat chloride concentration (SCC).
Methods: We retrieved 9-year longitudinal clinical data from the Dutch CF Registry of 173 people with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individual CFTR function was defined by FIS, measured as the relative size increase of intestinal organoids after stimulation with 0.8 µM forskolin, quantified as area under the curve (AUC). We used linear mixed effect models and multivariable logistic regression to estimate the association of FIS with long-term FEV1pp decline and development of pancreatic insufficiency, CF-related liver disease and diabetes. Within these models, FIS was compared with SCC.
Results: FIS was strongly associated with longitudinal changes of lung function, with an estimated difference in annual FEV1pp decline of 0.32% (95%CI: 0.11%-0.54%; p=0.004) per 1000-points change in AUC. Moreover, increasing FIS levels were associated with lower odds of developing pancreatic insufficiency (adjusted OR: 0.18, 95%CI: 0.07-0.46, p<0.001), CF-related liver disease (adjusted OR: 0.18, 95%CI: 0.06-0.54, p=0.002) and diabetes (adjusted OR: 0.34, 95%CI: 0.12-0.97, p=0.044). These associations were absent for SCC.

Conclusion: This study exemplifies the prognostic value of a PDO-based biomarker within a clinical setting, which is especially important for people carrying rare CFTR mutations with unclear clinical consequences.

Dr Danya Muilwijk is a PhD candidate in the Department of Pediatric Respiratory Medicine, Wilhelmina Children’s Hospital, University Medical Center, Utrecht University, Utrecht, The Netherlands.

Eyleen de Poel  is in the Department of Regenerative Medicine Utrecht, University Medical Center, Utrecht University, Utrecht, The Netherlands.

B L Aalbers, J E Brunsveld, C K van der Ent, J C van den Eijnden, J M Beekman, H G M Heijerman. Forskolin induced swelling (FIS) assay in intestinal organoids to guide eligibility for compassionate use treatment in a CF patient with a rare genotype. J Cyst Fibros 2022 Jan 30;S1569-1993(22)00028-5.doi: 10.1016/j.jcf.2022.01.008. Online ahead of print. [Pubmed]

Background: Forskolin-induced swelling of patient-derived organoids has been used to measure patient-specific CFTR function and CFTR modulator response. We present a case where CFTR function assessment in intestinal organoids was decisive for a patients’ acceptance to a compassionate use program.
Case description: A 56 years old female with cystic fibrosis compound heterozygous for F508del and a rare CFTR allele (c.3717+5G>T) experienced rapid clinical deterioration. The forskolin-induced swelling assay on her rectal organoids was used to confirm that the rare mutation is a minimal residual function mutation, and that other CFTR modulators would not likely be effective. Based on these two criteria and her clinical status, she was accepted for compassionate use of elexacaftor/tezacaftor/ivacaftor and showed improvement in all clinical parameters.
Conclusions: This reports describes a first example that intestinal organoids were used to identify a previously unknown CFTR mutation as a minimal function mutation. The individual FIS-based definition of minimal residual function, response to ele/tez/iva and/or lack of response to other CFTR modulating drugs, may thus provide a tool for access to ele/tez/ivatreatment for people with rare genotypes.

B L Aalbers  is in the Department of pulmonology, UMC Utrecht, The Netherlands. Electronic address: b.l.aalbers-2@umcutrecht.nl.

Marcel J C Bijvelds , Floris J M Roos  Kelly F Meijsen, Henk P Roest, Monique M A Verstegen, Hettie M Janssens, Luc J W van der Laan Hugo R de Jonge.  Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers.  J Cyst Fibros 2022 May;21(3):537-543.doi: 10.1016/j.jcf.2021.12.006. Epub 2021 Dec 23.  Free articl [Pubmed]

Background: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport.
Methods: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique.
Results: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation.

Conclusions: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

Marcel J C Bijvelds is a Principal Investigator in the Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center Rotterdam, PO Box 2040, 3000CA Rotterdam, the Netherlands.