- Titel
- Lung lineage induction through small molecules and partial cell reprogramming for lung organoid generation
- Untertitel
- Mimicking the pulmonary niche in vitro
- Zitierfähige Url:
- https://nbn-resolving.org/urn:nbn:de:bsz:15-qucosa2-987689
- Datum der Einreichung
- 02.02.2025
- Datum der Verteidigung
- 18.06.2025
- Abstract (EN)
- Prematurity-associated pulmonary complications pose significant challenges to both affected newborns and the healthcare system. To better understand the underlying causes of immature lungs and find effective treatments, there is a need for new in vitro and in vivo models. An accurate in vitro lung model should closely resemble the distal lung in terms of cell composition, structure, and function. Unfortunately, current lung models are not sufficient for studying the mechanisms of late lung maturation and developmental stages of pulmonary embryology. To address this need, research has turned to lung organoids as a promising tool. These 3D cell culture models, made up of various self-organizing progenitor or stem cells, closely replicate the structure of the corresponding organ and can be used to study lung maturation and disease processes. So far, organoids were derived from isolated organ progenitor cells or induced Pluripotent Stem Cells (iPSCs), and Embryonic Stem Cells (ESCs). However, logistical and ethical barriers currently hamper access to the corresponding tissue and prevent widespread implementation. To overcome these challenges, this study developed a lung model from progenitor and stem cells obtained from the placenta and umbilical cord. These tissues are an accessible and innovative source of stem cells, as they are usually discarded postnatally and do not raise ethical concerns. Epithelial progenitor cells (EpiPCs) were isolated from the amniotic epithelium, which already expresses lung progenitor cell markers, and endothelial cells (HUVECs) were isolated from the umbilical vein, while mesenchymal stem cells (hWJ-MSCs) were isolated from the Wharton's jelly. Subsequently, by co-culturing EpiPCs—without any prior induction toward lung lineage—with HUVECs, we were able to generate primitive human Lung Organoids (phLOs). To induce lung lineage in these phLOs, biophysical and biochemical cues were utilized to mimic the pulmonary niche. This included ALI-cell culture conditions, induced cell aggregation, and the use of an extracellular matrix. These techniques facilitated the more effective spreading of phLOs, leading to the formation of cell aggregates with sufficient space for further three-dimensional growth. As a result, a more uniform cell distribution, enhanced reproducibility, and alveolar-like growth could be observed, which further encouraged the development of lung lineages. Long-term culture of phLOs (phLOsDIV120) was also performed to test if lung-related gene expression (αENaC, βENaC, γENaC, SFTPB, SFTPC, CFTR) and alveolar-like growth patterns were enhanced over time. The findings indicated that lung-related gene expression increased significantly until DIV46 while no significant increase was observed after DIV120. In the long-term culture of phLOsDIV120 two growth patterns were observed: a cystic growth pattern with grape-like formations and delineated luminal structures and a branched growth pattern with condensed antler-like structures with an opaque appearance. Inducing lung lineage properties was found to be crucial not only for the final construct of phLOs but also, more importantly, for the initial cell populations. The closer these starting cells are to the in vivo lung environment, the more accurate the resulting lung model will be. Consequently, this study distinguishes between primitive hLOs (phLOs) and optimized hLOs. Unlike phLOs, optimized hLOs are derived from a starting cell population that has been induced towards lung lineage. The starting cells used to generate optimized hLOs include EpiPCs that have been manipulated with small molecules, as well hWJ-MSCs that have been manipulated through partial reprogramming. Lung lineage induction in EpiPCs involved mimicking the steps of lung development in vitro by activating signaling cascades including FGF, WNT, and SHH pathways through the use of small molecules. The addition of these small molecules to the EpiPCs culture medium aimed to increase the number of EpiPCs, while also inducing distal lung cell characteristics, primarily the induction of the lung fate transcription factor TTF-1 and the expression of lung-related genes such as CFTR, NKX2.1, SFTPB, SFTPC, and ENaC α/β/γ. The functions of ENaC and CFTR are important in maintaining fluid homeostasis in the lung and its adaptation to air breathing. AT2 cells synthesize and secrete surfactant, which prevents alveolar collapse during exhalation. NKX2.1 plays a crucial role in lung embryology, as its presence determines the respiratory lineage of endodermal cells. TTF-1 is the transcription factor of NKX2.1 and thus plays a vital role as a lineage-survival factor in lung development. All small molecules tested, including A83-01, CHIR99021, SAG, and FGF2, demonstrated a tendency to enhance the yield of EpiPC cells. However, only the application of the small molecule FGF2, as well as the combination of the SHH-Activator SAG and FGF2, led to a significant increase in the EpiPC cell count. While the WNT-Activator caused a notable decrease in TTF-1 expression, no significant effects on TTF-1 expression were observed from the other small molecules. All cultured EpiPCs exposed to small molecules exhibited the expression of lung-related genes, including αENaC, βENaC, γENaC, SFTPB, SFTPC, and CFTR. The SHH activator SAG significantly increased the expression of βENaC, while the WNT activator CHIR99021 significantly decreased the expression of αENaC. Additionally, the small molecule FGF2 notably reduced the expression of NKX2.1. HWJ-MSCs show great promise as an alternative cell population to EpiPCs that can be used to generate optimized hLOs. These cells offer high plasticity and are easily manipulable and isolated. To induce lung lineage, hWJ-MSCs require a process called mesenchymal-epithelial transition (MET), as the distal lung mainly comprises epithelial cells. MET can be induced through partial cell reprogramming, which involves introducing a cocktail of four pluripotency genes, namely NANOG, OCT4, KLF4, and SOX2 (NOKS), that are known to be sufficient for reprogramming somatic cells into pluripotent cells. During the initial stage of cell reprogramming, an alteration in the cell's expression pattern occurs, leading to epithelialization of the cells and paving the way for further lung lineage induction. To induce pluripotency gene expression and MET, retroviral transduction and mRNA transfection of hWJ-MSCs were utilized. Partially reprogrammed hWJ-MSCsNOKS showed significant expression of the pluripotency gene NANOG. However, there was no expression observed for the pluripotency genes OCT4 and SOX2, nor for the stem cell marker CXCR4. Half of the partially reprogrammed hWJ-MSCsNOKS successfully expressed the epithelial marker E-Cadherin, but EpCAM expression was absent. Successful cell reprogramming was demonstrated by changes in cell morphology characterized by the formation of tightly packed cell colonies resembling iPSCs, along with the loss of adherence. Both mRNA transfection and retroviral transduction led to the hWJ-MSCsNOKS adopting a rounded morphology and forming compact colonies with distinct borders and well-defined edges. These morphological changes indicated the epithelialization of the hWJ-MSCsNOKS and were interpreted as MET. Consequently, both gene transfer methods in this work effectively induced MET. After dramatic changes of the partially reprogrammed hWJ-MSCsNOKS in the sense of MET were observed, further lung lineage induction was conducted. hWJ-MSCsNOKS were additionally transfected with lung fate transcription factor TTF-1 mRNA and exposed to cell culture conditions promoting lung cell differentiation. Ultimately, it was shown that after additional transfection most partially reprogrammed hWJ-MSCsNOKS expressed the lung fate marker TTF-1, which is a crucial step for lung lineage induction. The objective of this work was to create a distal lung model that mimics the in vivo cell niche, promoting the study of fetal lung development, its perturbations, and therapeutic solutions. To achieve this, placental and umbilical cord progenitor and stem cells, representing a novel approach to generating hLOs were utilized. These cells were manipulated to exhibit lung lineage characteristics, resulting in an optimized distal lung model. Thereby this study aimed to contribute to future translational medicine applications, overcoming the limitations of current approaches to modeling human alveoli.
- Freie Schlagwörter (EN)
- Lung development, Lung organoids, Stem cell biology, Regenerative medicine
- Klassifikation (DDC)
- 610
- Den akademischen Grad verleihende / prüfende Institution
- Universität Leipzig, Leipzig
- Version / Begutachtungsstatus
- angenommene Version / Postprint / Autorenversion
- URN Qucosa
- urn:nbn:de:bsz:15-qucosa2-987689
- Veröffentlichungsdatum Qucosa
- 29.08.2025
- Dokumenttyp
- Dissertation
- Sprache des Dokumentes
- Englisch
- Lizenz / Rechtehinweis
CC BY-ND 4.0