Lung stem cells and respiratory epithelial chimerism in transplantation

Abstract

Stem cells are capable of self-renewal and differentiation into specialised types. They range from totipotent cells to multipotent or somatic stem cells and ultimately to unipotent cells. Some adult multipotent stem cells can have the potential to regenerate and colonise diverse tissues. The respiratory airways and lung mucosa, exposed to ambient air, perform vital roles for all human tissues and organs. They serve as barriers against airborne threats and are essential for tissue oxygenation. Despite low steady-state turnover, lungs are vulnerable to injuries and diseases from environmental exposure. Lung stem cells are crucial due to their regenerative potential and ability to replace damaged cells. Lung repair with extrapulmonary stem cells can occur, leading to the coexistence of respiratory cells with different genetic origins, a phenomenon known as airway epithelial chimerism. The impact of such chimerism in lung repair and disease is actively studied. This review explores different stem cell types, focusing on pulmonary stem cells. It discusses airway epithelium models derived from stem cells for studying lung diseases and examines lung chimerism, particularly in lung transplantation and haematopoietic stem cell transplantation, highlighting its significance in understanding tissue repair and chimerism-mediated repair processes in lung pathology.

Shareable abstract

Respiratory stem cells vary in roles and structure, impacting tissue repair and pathology. Epithelial chimerism, involving diverse origins, adds complexity. Ex vivo research could revolutionise personalised lung disease therapies. https://bit.ly/3D2arzo

Introduction

In the late nineteenth century, the term “stem cell” emerged in embryology and haematology [1]. Initially used to describe primordial germ cells, the term now broadly encompasses foundational cells capable of self-renewal and differentiation into specialised cell types (figure 1). Stem cells progress from totipotent cells, which appear immediately after fertilisation and have the potential to form any cell type, to multipotent or somatic stem cells that can differentiate only into different cell types present in the tissue of their origin and eventually unipotent cells, with decreasing differentiation potential at each stage. Recent studies have challenged the traditional view that adult multipotent stem cells are limited to producing a single type of differentiated cell. Evidence now shows that these cells can generate various cell types, including those from different germ layers, such as bone, heart muscle and lung cells. This discovery highlights the remarkable ability of stem cells to regenerate and colonise tissues beyond their original source [2]. Lung stem cells, specifically, are a significant focus of research due to their potential in regenerative medicine. These cells offer promising avenues for repairing and regenerating lung tissue, which can be crucial in treating respiratory diseases. Induced pluripotent stem cells (iPSCs), which are created by reprogramming mature cells into an embryonic-like pluripotent state, further expand the possibilities. iPSCs can develop into an unlimited source of any human cell type, providing invaluable potential for therapeutic applications, including lung regeneration.

FIGURE 1.

Schematic representation of human stem cells repartition and differentiation potency. Totipotent stem cell: a type of highly potent stem cell able to give rise to a fully functional, living organism. They can differentiate into embryonic tissue and the placenta. The best-known example of a totipotent stem cell is a fertilised egg (formed when a spermatozoid and egg unite to form a zygote). Around 4 days after fertilisation, these cells start specialising into pluripotent cells with diverse but limited differentiation potential. Pluripotent stem cell: a type of stem cell found in the inner cell mass of a mammalian blastocyst (embryonic stem cell) and capable of differentiating into the three germ layers: ectoderm, endoderm and mesoderm. These germ layers further differentiate to form all tissues and organs (examples are depicted). Induced pluripotent stem cell: a type of tissue-specific stem cell that can be reprogrammed to become functionally similar to embryonic stem cells. Multipotent stem cell (somatic stem cell): a type of stem cell able to self-renew and differentiate into a specific range of cell types. Examples: mesenchymal stem cells found in the mesenchymal stroma or connective tissues. They can differentiate into osteoblasts, myocytes, adipocytes and chondrocytes. Haematopoietic stem cells found in the bone marrow, peripheral blood and, to a lesser extent, umbilical cord blood of newborns. They can differentiate into lymphoid and myeloid progenitors that give rise to all blood cells and less frequently have the capacity to give rise to pluripotent cells (dashed arrow). Myeloid cells include basophils, dendritic cells, eosinophils, erythrocytes, macrophages, megakaryocytes, monocytes, neutrophils and platelets, while lymphoid cells include B-cells, T-cells and natural killer cells. Unipotent stem cell: a type of stem cell able to self-renew and differentiate into a single cell type. Only few unipotent stem cells are depicted but lung stem cells are an example. Figure created with BioRender.

The respiratory airways play a pivotal role as they are in contact with ambient air and the lung mucosa and serve as the primary interface with the external environment, acting as a barrier against airborne threats and toxins. Most of the time, the lungs are quiescent, with a low steady-state turnover rate. However, this vital organ is susceptible to various injuries and diseases due to its constant exposure to environmental factors. Once injured, the lung's ability to repair and regenerate is crucial for maintaining respiratory function and overall health. In normal lungs, different stem cells are present throughout each region [3, 4] (figure 2). These cells divide to replace old or damaged lung cells, which keeps the lung healthy [5–7] being able to renew the structure of the lung throughout normal adult life. The role of extrapulmonary stem cells in lung repair is still under investigation [8].

FIGURE 2.

Schematic representation of lung epithelial and/or stem cells along the airway. Schematic drawings of the lung anatomy with major cell types and stem cells. The airway epithelium is a continuous cell layer that mainly consists of epithelial cells (ciliary cells, basal cells, goblet cells and club cells) and pulmonary neuroendocrine cells (PNECs). Basal stem cells and club cells can differentiate into secretory cells and goblet cells. Goblet cells can differentiate into ciliated cells. Basal cells can also give rise to PNEC. Club cells and PNECs have the ability to self-renew. Club-like lineage-negative epithelial progenitors (LNEPs) are multipotent stem cells that can either give rise to club cells or alveolar cells. Alveoli are the smallest passageway in the respiratory system, which consist of alveolar type (AT) 1 and 2 cells. AT2 can self-renew and give rise to AT1. Both AT1 and AT2 can also arise directly from a bipotent progenitor. BASC: bronchioalveolar stem cell. Figure created with BioRender.

This understanding of stem cell plasticity also introduces the concept of chimerism in transplantation, where donor-derived stem cells can integrate and function within the recipient's tissue.

Chimerism designates the presence of genetically distinct cell populations within a single organism. Airway epithelial chimerism describes the coexistence of respiratory cells with different genetic origins within the airway epithelial layer. The potential contribution of airway epithelial chimerism to lung repair processes and lung disease is an area of active study.

This review provides an overview of different stem cell types, with a specific focus on pulmonary stem cells and their role in lung pathology. It also discusses various models of airway epithelium, including those derived from stem cells for studying lung diseases. The state of knowledge on lung chimerism is also discussed, particularly in relation to lung transplantation (LT) and haematopoietic stem cell transplantation (HSCT), highlighting its significance in understanding tissue repair mechanisms in lung pathology, particularly in the context of stem cell differentiation and chimerism-mediated repair processes.

Airway epithelial cells and stem cells

The respiratory epithelium significantly differs along the proximal distal axis both in epithelial and stem cell composition and proportion (figure 2). The complex structure of the lungs is made up of approximately 58 cell types [9]. During the branching of the bronchi into bronchioles and terminal bronchioles, the epithelium transitions from pseudostratified to simple cuboidal. This change is accompanied by a gradual decrease in the number of ciliated, goblet and basal cells, while club cells become the predominant cell type. The main epithelial cell types of the respiratory tract and the somatic lung stem cells are briefly described. Several of them can serve as unipotent progenitor of respiratory epithelial cells along the airway. It has also been shown that, in fully developed lungs, some populations of multipotent endogenous stem cells located in niches can give rise to different airway epithelial cell types. Finally, circulating progenitor epithelial cells coming from the bone marrow can contribute to re-epithelialisation of the airway and re-establishment of the pseudostratified epithelium in case of injury [10].

Respiratory cells and stem cells

The different cells lining the human respiratory tract are briefly described and the common markers of these cell are summarised in table 1. Cells with no stem cell potential are highlighted with an asterisk in the following sections.

TABLE 1.

Common markers of airway epithelial cells

Cell type	Markers
Basal cells	KRT5, KRT14, TP63
Goblet cells	MUC5AC, MUC5B
Club cells	SCGB1A1, CC10, CCSP
Ciliated cells	CHDR3, FOXJ1
LNEP	H2-K1
BASC	CCSP, SFTPC
AT1	AQP5, HOPX, AGER
AT2	SFTPC, SFTPB, NAPSA, ABCA3, LAMP3
Tuft cells	TRMP5, DCLK1, POU2F3
Ionocytes	CFTR, FOXJ1, ASCL3, STAP1
PNEC	CHGA, CGRP

ABCA3: ATP binding cassette 3; AGER: advanced glycosylation end product-specific receptor; AQP5: aquaporin 5; ASCL3: achaete-scute family BHLH transcription factor 3; AT: alveolar type; BASC: bronchioalveolar stem cell; CC10: club cell 10; CCSP: club cell specific protein; CFTR: cystic fibrosis transmembrane conductance regulator; CGRP: calcitonin gene-related peptide; CHDR3: cadherin-related family member 3; CHGA: chromogranin A; DCLK1: doublecortin-like kinase 1; FOXJ1: forkhead box J1; H2-K1: histocompatibility 2, K1 region; HOPX: homeodomain protein X; KRT5/14: keratin 5/14; LAMP3: lysosomal associated membrane protein 3; LNEP: lineage-negative epithelial progenitor; MUC5AC/5B: mucin 5AC/5B; NAPSA: napsin A; PNEC: pulmonary neuroendocrine cell; POU2F3: POU class 2 homeobox 3; SCGB1A1: secretoglobin family 1A member 1; SFTPB/C: surfactant associated protein B/C; STAP1: signal transducing adaptor family member 1; TP63: tumour protein 63; TRMP5: transient receptor potential melastatin 5 channel.

Basal cells

The distribution of basal cells in the airway epithelium follows a pattern, with the highest proportion observed in the large airways and decreasing progressively along the tracheobronchial tree. Specifically, the trachea exhibits an average of 34% basal cells, the large airways 27% and the small airways 10% [11]. Airway basal stem cells (BSCs) and subpopulations of BSCs express varying cell markers depending on their spatial location in the airways [12, 13]. Under homeostatic conditions, BSCs can differentiate into secretory, ciliated and neuroendocrine cells while retaining self-renewal capacity [14]. Isolated from adult tissues, they can be used to generate fully differentiated airway epithelia ex vivo [15]. In response to acute injuries, BSCs rapidly go from a quiescent state towards proliferation and differentiation to restore epithelial homeostasis [15]. Ongoing studies aim to elucidate molecular mechanisms regulating BSC progenitor properties and their role in airway epithelial homeostasis and repair, with implications in airway remodelling observed in diseases such as COPD, genetic diseases such as cystic fibrosis (CF), asthma, idiopathic pulmonary fibrosis (IPF) and lung squamous cell carcinoma [16–22].

Secretory cells

Goblet cells, so named because they are shaped like a wine goblet, are columnar epithelial cells that contain mucous granules. They are situated in the epithelium of the conducting airways from the trachea to the larger bronchioles, often with their apical surfaces protruding into the lumen. Together with the submucosal glands, goblet cells secrete gel-forming mucins, such as mucin 5AC, as part of the airway lining fluid. This thick layer is important to trap microbes at the apical side of the respiratory epithelium preventing infection of the underlying epithelial cells. Under normal conditions, the proliferation and differentiation of goblet cells, into ciliated cells, work together with basal cells to sustain the airway epithelial cell population [23].

Club cells constitute approximately 9% of the total population of airway epithelial cells in human lungs [24]. Their proportion in the terminal and respiratory bronchioles is respectively 11% and 22%, and they are practically absent in the mucosal barrier of the proximal segments of the bronchial tree (the trachea and the primary, lobar and segmental bronchi) [25]. Club cells play a vital role in secreting surfactants (such as surfactant proteins A, B and D) and other proteins (such as club cell secretory protein, also known as secretoglobin 1A1) that contribute to the airway lining fluid. This surfactant plays, among others, a defence role similar to the mucus in the lower airways where goblet cells are rarer or absent. Recent evidence has also indicated that club cells are able to differentiate into alveolar type (AT)-2 and 1 during the restoration of the damaged alveolar epithelium [26]. Variants of club cells serve as epithelial progenitors in small airways following injury, contributing to epithelial regeneration through self-renewal and differentiation into ciliated cells [27].

Lineage-negative epithelial progenitors (LNEPs)

In instances of lung injury, recent research identified a specific subgroup (approximately 5%) of LNEPs that are present within normal distal lung. These LNEPs are crucial for alveolar repair [28, 29]. They present proliferative capacity and multipotency, contributing to alveolar regeneration but also restoration of club cells in case of injury [28].

Ciliated cells*

Ciliated cells are columnar epithelial cells with specialised ciliary modifications but without known stem cell potency. They are descendants of basal cells and also of club cells or goblet cells and make up between 10% and 50% of the epithelium depending on the site of the respiratory tract, except in the alveoli where they are absent [30]. Ciliated cells are responsible for displacement of mucus and trapped microbes towards the digestive tract or the upper respiratory tract allowing their elimination via swallowing, sneezing or coughing. They are key players of the mucociliary clearance mechanism. Each epithelial cell has around 200 cilia that beat constantly at a rate between 10 and 20 times·s⁻¹ [31].

Bronchioalveolar stem cells (BASCs)

BASCs, proposed as unique endogenous stem cells located at bronchioalveolar–duct junctions for lung regeneration, have sparked debate regarding their in vivo role and true contribution to lung repair [32]. Using a mouse model, BASCs were tracked in vivo, revealing their response to lung injuries and ability to differentiate into diverse cell types, aiding lung regeneration [33]. These recent investigations, as well as human studies utilising single-cell RNA-sequencing analysis [28, 34], have confirmed the phenotypic lineage plasticity of BASCs and their ability to transdifferentiate into basal cells and various other cell types. These findings underscore the multipotent role of BASCs in lung repair and regeneration, reshaping our understanding of cellular dynamics in lung regeneration.

Alveolar cells

AT1 cells^⋆: AT1 are complex, highly branched cells characterised by thin cytoplasmic plates that form the primary gas exchange surface within the alveolus. They are relatively devoid of organelles to facilitate efficient gas diffusion.

AT2 cells: AT2 maintain the integrity and the function of the alveolar space. Additionally, AT2 cells synthesise, store and release pulmonary surfactant into the alveolar space, optimising conditions for gas exchange.

More than 99% of the lung internal surface area is located within the alveolar compartment, which is lined by two types of cells: AT1 and AT2 cells. AT1 cells constitute about 8% of the lung's total cells but cover approximately 95–98% of the surface area, while AT2 cells make up around 14% of the lung's cells and account for the remaining 2–5% of the internal surface area [35].

Previous data suggested that AT1 arise from AT2 cells, but recent studies propose other origins for AT1 cells [36]. In their study, Desai et al. [37] showed that during development, AT1 and AT2 cells arise directly from an unnamed bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. AT2 normally exhibit quiescence with a slow turnover rate [37], but they demonstrate remarkable regenerative capabilities when activated upon injuries. Studies have shown that AT2 cells possess clonal growth potential in vitro [38], functioning as alveolar progenitors and long-term stem cells in adult lungs, with support from neighbouring stromal cells such as lipofibroblasts [39]. Accordingly, AT2 cells can play a role as pathological or beneficial stem cells in various lung pathologies, including infections, cancer and fibrosis. In IPF, an accumulation of cells resembling the transitional state between AT2 and AT1 cells may impede alveolar regeneration, potentially contributing to fibrosis.

Other rare cell types*

Tuft cells are characterised by their distinctive microvillous apical tuft and are found in various tissues (gastrointestinal tract, airways, thymus). They are normally found in the trachea and bronchi and under pathophysiological conditions, they have been detected in the alveoli of children [40]. Tuft cells are believed to have chemosensory, neuronal and immunological functions. Pulmonary ionocytes are a major source of CF transmembrane conductance regulator activity in the conducting airway epithelium that contribute to the regulation of surface liquid pH [41].

Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells that also uniquely harbour neuronal and endocrine characteristics. Located close to immune cells and nerves, PNECs play a central role in the lungs' perception of and response to the environment [42].

Despite emerging evidence implicating these cells in respiratory diseases, much about their multifaceted functions remains to be uncovered.

Nonrespiratory stem cells able to differentiate into respiratory cells

Mesenchymal stromal/stem cells (MSCs)

MSCs are multipotent cells first isolated from bone marrow and found in the mesenchymal stroma or connective tissues of the body including lung tissue [43–45]. Lung-resident MSC (LR-MSCs) encompass a variety of cell types, including resident fibroblasts, lipofibroblasts, myofibroblasts, smooth muscle cells and pericytes, which all occupy different anatomic locations and exhibit diverse homeostatic functions in the lung. Accordingly, they play important roles in maintaining pulmonary homeostasis by modulating immune responses, releasing antifibrotic factors and contributing to lung tissue integrity [46]. While endogenous MSCs are involved in lung repair, they can also contribute to cellular senescence and tissue aging, potentially affecting lung regenerative capacity. LR-MSCs can exacerbate lung diseases through altered secretory and immunomodulatory properties, as well as by differentiating into myofibroblasts, promoting fibrotic lung diseases [47–49]. The multifaceted role of LR-MSC underscores their impact on lung homeostasis and pathological conditions, highlighting their potential as therapeutic targets for lung diseases.

Endothelial progenitor cells (EPCs)

Pulmonary EPCs can be found as resident in the lungs or circulating in the vascular system. The lack of distinct markers for these two EPC populations makes it challenging to determine their physiological functions and role in tissue reconstruction. Their limited numbers add difficulty in understanding their regenerative potential. Nonetheless, studies have shown their direct proliferative capacity in lung endothelial regeneration [3, 50].

Stem cells and injury

Repair capacity following injury varies from one organ to another. Scarring or sometime loss of function are mechanisms involved after injury. Chronic and acute lung diseases are complex mechanisms that may be linked to defective repair and involve a multitude of epithelial cell types.

Respiratory stem cells are one of the multiple contributors to lung repair. Stem cells exhibit a dual role in pathology, functioning both as protective factors, with their absence contributing indirectly to pathological processes or being themselves potential sources of disease. The involvement of stem cells is described below in some conditions including pulmonary infections, lung fibrosis, COPD, genetic diseases such as CF and asthma, pulmonary arterial hypertension, and transplantation. Both viral infection and chronic lung disease share common features, including stem cell loss in alveoli, basal cell hyperplasia in small airways and innate immune activation, contributing to epithelial remodelling and loss of lung function. Due to their properties of self-renewal and proliferation, stem cells can survive chemotherapy and radiotherapy and can thus be involved in the development and progression of lung cancer [51]. Though ciliated cells do not have the ability to proliferate or transdifferentiate, they can transiently change their morphology in response to lung injury [52, 53]. A summary of the main respiratory disease and the different respiratory stem cells involved is given in table 2 [47, 51, 54–65].

TABLE 2.

Stem cell (SC) involvement in the main respiratory diseases

Lung disease	Type of SC	SC characteristics	References
Lung cancer	Basal SC Club SC PNEC	Uncontrolled growth and altered genotypes Resistance to therapy (chemo/radiotherapy)	[14, 18, 19, 24, 49–52, 54]
Respiratory viral infection	Basal SC BASC	Increase virus infection, production and transmission	[27, 55–57]
COPD	Basal SC BASC, AT1 and AT2 Submucosal bronchial SC LR-MSC	Basal SC exhaustion Epithelial expansion Mucous secretory cell hyperplasia/metaplasia Mucus accumulation Pro-fibrotic phenotype of LR-MSC	[17, 20, 58]
Lung fibrosis	Basal SC AT1- AT2 Fibroblast SC LR-MSC	Exhaustion resident SC Loss of epithelial progenitor cells AT2: dysregulation of repair and hyperplasia Accumulation of fibroblasts Stroma's uncontrolled proliferation	[16, 21, 44, 63]
Asthma	Airway smooth muscle SC Goblet cell Endothelial SC Lung fibroblast SC LR-MSC	Airway wall thickening Metaplasia of goblet cells Increased mucus production and loss of cilia Angiogenesis in airway wall fibrosis	[27, 43, 59–62]
Cystic fibrosis	All airway epithelial SC variants	Increase and drive neutrophilic inflammation Long-term proliferative potential (independent of CFTR activity)	[22, 24, 64]
Pulmonary hypertension	Endothelial SC Smooth muscle cells LR-MSC (fibroblasts and pericytes)	Perivascular inflammation Smooth muscle cell accumulation Intimal thickening Adventitial fibrosis	[27, 65]

AT1/2: alveolar type 1/2; BASC: bronchioalveolar SC; CFTR: cystic fibrosis transmembrane conductance regulator; LR-MSC: lung-resident mesenchymal SC; PNEC: pulmonary neuroendocrine cell.

Chimerism

Definition

The term chimerism dates back to the 1970s. It refers to chimera which is, in Greek mythology, a fire-breathing female monster resembling a lion in the forepart, a goat in the middle and a dragon behind [66]. In biology, the first observation of chimerism was reported in 1945 by Owen [67]. It is defined by the presence of more than one genetically distinct population of cells in a single organism that originated from more than one zygote.

Microchimerism

Microchimerism occurs when the nonhost cells represent only <1% of the cells of an individual [68]. The main situation of natural microchimerism is pregnancy where a bidirectional cell trafficking between mother and fetus is present. This exchange of cells can lead to a microchimerism in both the mother and child that can persist long after the birth and the consequences are still not well known. Some data support that it might be involved in the development of selected autoimmune disease in the mother [69–71]. Cells from these microchimeric tissues may act as direct effectors responding to maternal major histocompatibility complex antigens, potentially boosting immune responses through indirect antigen presentation [72]. For example, the human leukocyte antigen (HLA) phenotype DQA1 050 was identified as a potential indicator of a higher risk of autoimmune disease linked with microchimerism [73].

Airway epithelial chimerism refers to the presence of respiratory cells with different genetic origins within the epithelial layer of the airways. Its impact may be positive or negative. For example, in adult women after childbearing age, hypersensitivity pneumonitis has been associated with increased fetal microchimerism [74]. On the contrary, microchimerism may offer a protective effect against childhood asthma rather than heightening the risks associated with maternal asthma [75]. Microchimerism was also investigated in respiratory infections, with a case–control study with a cohort of 64 COVID-19 patients. Among infected women, the presence of fetal microchimerism did not significantly affect disease severity, but all deceased women were fetal microchimerism negative, speaking in favour of an association between microchimerism and positive outcome [76]. A study involving a murine model of cytomegalovirus infection has similarly highlighted that reduced levels of maternal microchimeric cells in the progeny increased the severity of viral infection [77]. Finally, fetal microchimeric cells expressing specific proteins are implicated in maternal tissue repair post-injury, suggesting a potential role in healing the injured respiratory epithelium [78]. Microchimerism might thus positively influence the healing process of an injured respiratory epithelium. A possible explanation being that the presence of genetically distinct cells within the lungs may influence the immune responses and contribute to the complex dynamics of lung-related disorders.

Chimerism

Chimerism in the setting of transplantation is defined by a higher proportion of nonhost cells (>1%). It can be detected in several types of solid organ transplantations and following HSCT. Depending on the transplantation, two different situations occur: the donor cells are identified outside the transplanted organ or blood in case of HSCT, or the recipient cells are identified in the transplanted organ for solid organ transplantation.

Detection of chimerism

The demonstration of chimerism relies on several techniques that allow to distinguish cells or tissues with different genetic origins within a single organism [79]. In case of sex mismatch between a donor and a recipient, cytogenetics such as fluorescence in situ hybridisation (FISH) can be used to distinguish between donor and recipient. Briefly, fluorescence enables the visualisation of the X and Y chromosomes in nuclei of interphase cells using probes which recognise repetitive sequences on the X and Y chromosomes [80].

In the case of solid-organ transplants, HLA compatibility may be partial [81]. Classically in this case, immunohistochemistry is used to detect chimerism to identify specific antigens of each individual. Chimerism can also be detected using blood group typing [82]. This can result in a number of situations, such as in pregnancy and HSCTs, with a group mismatch between donor and recipient. Blood group, if one of the individual lacks a given antigen, is determined by real-time quantitative PCR on cell-free DNA [83].

Nowadays, genetic markers can be identified using microsatellite analysis, detection of single nucleotide polymorphisms or short tandem repeat profiles of DNA, most often in noncoding regions. More recently, the use of digital PCR, next-generation sequencing (NGS) and single-cell NGS allows genotyping and monitoring of chimerism with high performance and sensitivity [84].

Chimerism and transplantation

In the context of transplantation, the notion of chimerism is closely linked to immunological tolerance.

Blood chimerism

In recipients following HSCT, chimerism can be categorised into complete and mixed chimerism. Complete chimerism occurs when the patient's entire haematopoietic system is replaced by donor cells, whereas mixed chimerism reflects the coexistence of both recipient and donor-derived blood cells. Complete blood chimerism is generally the goal, as early levels of donor chimerism can predict relapse and survival rates post-transplantation [85]. Long-lasting donor blood chimerism is also an indicator of post-HSCT tolerance [86, 87]. Some experts propose that mixed chimerism could be beneficial, potentially leading to tolerance through the deletion of donor and recipient reactive cells in the thymus via negative selection [88]. Attention has shifted towards chimerism in specific haematopoietic cell lineages, particularly those relevant to graft-versus-host disease (GvHD) and graft-versus-leukaemia effects, including T-lymphocytes, dendritic cells and natural killer cells. These lineages play crucial roles in both complications and therapeutic benefits post-HSCT. However, the implications of mixed chimerism for engraftment and GvHD remain controversial and warrant further investigation [89–91].

Post-HSCT, endothelial cells and myofibroblasts in the marrow normally originate from the donor. A loss of chimerism and the persistence of host cells in the medullary endothelium was associated with relapse of the haematological disease [92].

Solid-organ transplant recipients: after solid-organ transplantation (such as intestinal or liver–intestinal grafts), donor-derived circulating mononuclear cells can transiently constitute 5–15% of the total circulating mononuclear cells in the early post-operative period [93].

After LT, it has been suggested that at 1-month post-surgery, a chimerism of more than 30% of donor lymphocytes or macrophages within the lung correlates with better graft tolerance [94]. Chimerism has also been described with repopulation of pneumocytes by bone marrow-derived stem cells of the recipient [95–97] and engraftment of donor cells in several other organs with a transient detection in the blood [98, 99].

Several studies have demonstrated that human chimerism of circulating leukocytes is linked to improved graft acceptance and reduced rejection rates following solid organ transplantation [86, 93, 100, 101]. Furthermore, a high level of donor chimerism in various immune cell types can lead to immunological tolerance and has been shown to allow the withdrawal of immunosuppressive medication [102]. Nevertheless, some clinical studies have not found a consistent effect of chimerism on heart, liver or kidney graft acceptance [103, 104]. In liver transplant cases, chimerism has even been observed alongside graft rejection [105, 106]. These discrepancies in clinical outcomes may stem from variations among patients in the quantity or types of chimeric cells present. It has been proposed that the specific type of chimeric cells could be a critical factor in determining whether chimerism is associated with graft tolerance or immunity against host-derived foreign cells [107].

Tissue chimerism

In HSCT recipients: beyond blood chimerism following HSCT, research has demonstrated chimerism in nonhaematopoietic cells derived from the donor in various recipient organs [108–112].

This phenomenon has been observed in animal studies and, more recently, in human studies, including the examination of lung specimens from allogeneic HSCT recipients [111, 113, 114]. In mice, chimerism has been shown to occur predominantly in the alveolar space following HSCT [115, 116].

In solid-organ transplant recipients, the cells observed within the transplanted organ are expected to be those of the donor. It may happen, after liver transplantation for example, that donor cells are also found in an organ other than the one transplanted [102]. This was first observed in liver transplant recipients [93]. In this study, donor cells were detected in the recipient skin, lymph nodes, small bowel and blood up to several years after liver transplantation [93]. Studies indicate two different phenomena: the presence of recipient-derived cells within the organ transplanted or the presence of donor-derived cells in other organs than the one transplanted. It is the result of the migration of stem cells from the allograft into the peripheral circulation and tissues of the recipient.

Chimerism has also been described in nonepithelial cells such as endothelial cells or myofibroblasts. It is generally believed that the endothelium in solid-organ transplantations remains of donor origin and is not replaced by that of the recipient [117]. However, studies of re-endothelialisation of the kidney, heart and liver after transplantation have shown that some of the endothelial cells are of recipient origin [118–122].

A single study demonstrated long-term respiratory chimerism (up to 3 years post LT) using FISH analysis for X and Y chromosomes in sequential transbronchial biopsies [97].

Lung cell transplantation is an emerging therapeutic approach aimed at regenerating damaged lung tissue by introducing donor-derived lung progenitor cells into injured lungs. This strategy seeks to restore function by integrating donor cells within the tissue of the host, forming a chimerism of both cell types [123]. Studies in mice have demonstrated that donor epithelial cells merge with host cells to aid lung repair, confirming its therapeutic potential. For example, transplantation of canalicular-stage embryonic lung cells into irradiated mice led to long-term lung chimerism, with donor cells contributing to epithelial, mesenchymal and endothelial tissues, significantly improving lung function [124]. In lung fibrosis models, donor lung progenitor cells integrated into damaged lungs without prior conditioning, reducing fibrosis and enhancing function [125]. Additionally, co-infusion of haematopoietic and lung progenitor cells enabled donor cell engraftment without immune suppression, highlighting a promising approach for lung regeneration [126].

Chimerism and chronic allograft lung dysfunction

Long-term survival after HSCT and LT is limited by chronic noninfectious lung dysfunction. This manifests as chronic lung GvHD, primarily bronchiolitis obliterans syndrome (BOS), after HSCT and chronic lung allograft dysfunction (CLAD) after LT. Despite differing underlying immunopathologies, both conditions result in similar structural lung remodelling and respiratory dysfunction. Risk factors include attacks on the bronchial epithelium through physical, chemical or microbial stresses [127–133].

The role of respiratory epithelial chimerism in these complications remains to be elucidated.

Studies on human lung allografts indicate higher chimerism in bronchi and damaged epithelial structures, suggesting extrapulmonary precursor cells contribute to pulmonary regeneration [134]. Chimeric cells in airway epithelium have been shown to prevent luminal airway fibrosis in mice and predict BOS in porcine models [135, 136]. Recipient-derived epithelium may provide protection against immunologic injury and promote allograft tolerance [137].

In CF lung transplants, resident tissue stem cells exhibit bidirectional migration, impacting ion homeostasis and host defence [138]. Low donor lymphocyte numbers and high donor alveolar macrophage numbers in transplanted lungs correlate with worse clinical outcomes and earlier BOS development [139]. This supports the hypothesis that donor lymphocytes' graft-versus-host reaction neutralises the host-versus-graft response, aiding graft acceptance [140].

Conversely, donor cell colonisation post-HSCT might be beneficial, as these cells have not undergone chemotherapy and may better repair epithelial injury. Exogenous progenitor cells can aid airway repair when resident cell niches are overwhelmed. Low chimerism could indicate a poor prognosis by promoting BOS. Studies show functional donor-derived AT2 cells expressing surfactant protein predominantly in alveolar regions, aligning with the regenerative potential of distal lung compartments [97, 108, 111, 112, 134, 141, 142].

However, the predictive value of airway epithelial chimerism remains unclear. No definitive relationship between tissue injury and chimerism has been established, partly due to limited patient samples and normal tissue scarcity in specimens. No correlation between epithelial or endothelial chimerism and active GvHD has been found [111, 143].

Currently, there is no animal model accurately reproducing lung GvHD or CLAD. These multifactorial complications require ex vivo models to vary each variable and improve understanding and treatment. Understanding respiratory chimerism dynamics could enhance tissue biology knowledge and inform regenerative medicine and therapeutic interventions.

Ex vivo modelling of bronchial epithelium and pulmonary chimerism

To study airway epithelium and chimerism effectively, reliance on murine models should be minimised. Human lungs exhibit distinct localisation of stem and progenitor cells compared to rodents, posing challenges in translating research findings to successful clinical applications and drug development despite initial success in rodent testing [144]. Standard two-dimensional monoculture cell models using immortalised human lung epithelial cell lines do not faithfully reflect physiological reality [145].

To address these challenges and ethical considerations, researchers are turning to more relevant ex vivo and in vitro models. Human pluripotent stem cells (hPSCs), including embryonic stem cells and iPSCs, offer a valuable resource for generating various lung cell types to study respiratory diseases, accelerate drug development, and explore personalised medicine and cell therapy [146–148] (figure 3).

FIGURE 3.

Schematic representation of the lung modelling for lung pathology. Immortalised epithelial cell lines, mesenchymal stem cells, basal stem cells obtained from ex vivo nasal or bronchial samples from patients or human induced pluripotent stem cells are different sources for lung modelling. These cells are used for in vitro two-dimensional (2D) and air–liquid interface culture models or organoids (3D). The model can be enhanced through co-culture systems with the addition of immune cells. It can also serve as a model of infection or to test drugs. Lung tissue availability can be leveraged in ex vivo lung tissue models using small sections, maintaining the native architecture. Tissue explants enable the development of precision-cut lung slices. Miniaturised models, such as lung-on-a-chip, integrate microfluidics, engineering and cell biology to recreate aspects of organ physiology in vitro, providing a promising alternative. Figure created with BioRender.

The air–liquid interface (ALI) culture system, employing primary or hPSC-derived cells (mainly basal cells), allows the development of polarised structures resembling native lung tissue with functional cilia and mucous secretion in contact with air, facilitating the study of lung biology, infectious diseases and drug screening [149–151].

Lung organoids, derived from adult or fetal lung stem cells and iPSCs, provide a unique platform to study lung physiology and diseases, model organ development and, as well as an ALI system, enable genetic manipulations [148, 152–159]. Microfluidic airway-on-a-chip devices mimic lung tissue interfaces and physiological conditions, featuring bronchiolar epithelium and microvascular endothelium, for studying respiratory diseases at various scales [160–163].

Bioprinting technology enables the creation of complex 3D lung models using cell-laden bioinks, showing promise in surgical simulation and integration with microfluidic systems to replicate in vivo environments [164–166].

Precision-cut lung slices preserve lung architecture and cell interactions, offering a standardised model for studying lung physiology and diseases, despite limitations in material availability and culture duration [167].

These ex vivo and in vitro lung models leverage human pluripotent stem cells and innovative culture systems to provide more clinically relevant and ethical approaches for studying airway epithelium and chimerism in respiratory diseases. They hold promise for advancing our understanding of disease mechanisms, drug discovery and personalised treatments without relying on murine models, especially for nonalveolar diseases. A Venn diagram represents the different respiratory models for lung pathology, considering ethical limitation, clinical relevance, availability and reproducibility (figure 3). The hierarchy is different when it comes to the development of drugs, where the throughput comes first for example.

Conclusion

Throughout the respiratory tract, the stem cells that give rise to epithelial cells differ in their role and structure. These cells play a role in tissue repair but may also lead to pathology.

In parallel, the emerging concept of chimerism in respiratory epithelial cells adds another layer of complexity, with its mechanisms and promoting factors yet to be elucidated. These lung chimeric cells can originate from respiratory or extra respiratory (haematopoietic, endothelial) sources. Furthermore, the involvement of this chimerism in post-transplant rejection phenomena, chronic lung disease and response to infections or injuries remains to be determined. A better understanding of the respective roles of stem cells and chimerism using appropriate ex vivo models might help predict and improve treatment of lung diseases in general and lung injury in lung transplant and HSCT recipients in particular. Such research on stem cell functions and chimerism might thus revolutionise respiratory medicine, offering more effective and personalised treatments for severe lung conditions.

Questions for future research

• What are the mechanisms underlying the integration of lung stem cells and extrapulmonary stem cells in tissue repair?

• How does airway epithelial chimerism influence long-term lung function and repair after transplantation?

• Future research addressing these questions will deepen our understanding of lung regeneration and improve therapeutic strategies for respiratory diseases.