key: cord-0818051-0qjo94ty
authors: Varma, Ratna; Soleas, John P.; Waddell, Thomas K.; Karoubi, Golnaz; McGuigan, Alison P.
title: Current strategies and opportunities to manufacture cells for modeling human lungs
date: 2020-08-22
journal: Adv Drug Deliv Rev
DOI: 10.1016/j.addr.2020.08.005
sha: 2a3d753e790abce94f2cc68bf329917f192d99bc
doc_id: 818051
cord_uid: 0qjo94ty

Chronic lung diseases remain major healthcare burdens, for which the only curative treatment is lung transplantation. In vitro human models are promising platforms for identifying and testing novel compounds to potentially decrease this burden. Directed differentiation of pluripotent stem cells is an important strategy to generate lung cells to create such models. Current lung directed differentiation protocols are limited as they do not 1) recapitulate the diversity of respiratory epithelium, 2) generate consistent or sufficient cell numbers for drug discovery platforms, and 3) establish the histologic tissue-level organization critical for modeling lung function. In this review, we describe how lung development has formed the basis for directed differentiation protocols, and discuss the utility of available protocols for lung epithelial cell generation and drug development. We further highlight tissue engineering strategies for manipulating biophysical signals during directed differentiation such that future protocols can recapitulate both chemical and physical cues present during lung development.

End-stage lung disease is the third leading cause of morbidity and mortality worldwide events that occur, that differentiation models attempt to mimic, and highlight how human lung embryology has served as the blueprint to create the common pathway of lung directed differentiation protocols. We then discuss the evolution of directed differentiation protocols to find opportunities for creating specific populations of airway and lung epithelia through targeted manipulation of key signaling pathways in 2D and 3D models. We further describe how these models have been used to recapitulate different airway and lung diseases. Finally, we discuss how tissue engineering and biophysical cues using biomaterials can be utilized during lung directed differentiation to mimic patterning cues present in development to augment current differentiation protocols.

Directed differentiation protocols have been designed to mimic in vivo human lung development [39] . Indeed, in vitro models of lung development have provided unique insight into human lung development [40] . As human lung development has been described at great length in earlier reviews, [41, 42] , we provide a brief overview as follows (schematically represented in Figure 1 ). During early embryogenesis (at 14 days post fertilization), a process called gastrulation begins with the appearance of a structure called primitive streak, through which cells migrate to form the primary embryonic germ layers (definitive endoderm, mesoderm, and ectoderm) [43] [44] [45] . Definitive endoderm expands, thereby forming the primitive gut tube comprised of three endodermal regions: foregut, midgut, and hindgut [46, 47] . This is when lung development begins, at approximately four weeks into embryonic life, with the outgrowth of foregut endoderm [48, 49] and continues through eight years of post-natal life [50] .

There are five stages to lung development:

1. Embryonic (weeks 4-7): The future lung buds emerge from the ventral side of the primitive foregut endoderm into the surrounding mesenchyme and develop into embryonic lung buds with early trachea and bronchi [51] [52] [53] [54] [55] . 3 . Canalicular (weeks [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] : Development of the respiratory, or gas-exchanging, airways is initiated, primitive alveoli form, and the future distal epithelium begins to thin as distal epithelial markers are expressed [41, 57] . 4 . Saccular (weeks [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] : Emergence of sac-shaped distal airways, which develop crests with muscle and elastin to create indentations. These distal airways extend to form alveoli by 29 weeks [58] . The developing epithelium and vasculature within the future alveolus continue to merge closer together to facilitate future gas exchange and further differentiation of alveolar epithelial cells (AEC) I and II takes place. Septation leads to an increase in surface area of the gas exchanging portion of the developing lung and prepares the fetus to breath air during this stage [50, 59] . 

During the embryonic period, early lung is genetically defined by the expression of transcription factor NK2 Homeobox 1 (NKX2.1) and Sry-box 2 (SOX2) [60] [61] [62] . During human lung development, it has been found that the lung buds and branches given off during the pseudoglandular period are mostly SOX2 + SOX9 + [17, 62, 63] . Both SOX2 and SOX9 are individual markers of the early proximal or distal lineage, respectively [60, [64] [65] [66] . Over the course of the canalicular and saccular periods of development (weeks , these double positive populations downregulate one SOX protein and maintain expression of the other as these cells mature towards proximal or distal lineages [62] . The proximal airway (closer to the mouth) is comprised of a pseudostratified columnar epithelium that is responsible for the conducting airway function: debris and pathogen removal (ciliated cells), mucus production (goblet cells), prevention of airway inflammation (club cells), and humidification of air as it passes through to the distal lung compartment [67] [68] [69] . The squamous distal epithelium, J o u r n a l P r e -p r o o f composed of alveolar epithelial cells (AEC I and II), facilitates the respiratory function of the lung as air in the epithelial compartment is brought into close apposition to blood from the pulmonary vasculature; it also secretes surfactants, which play an immunologic role and decrease the surface tension present at the air-liquid interface, thereby preventing alveolar collapse [70] .

In humans, a number of cell types are found in the proximal airway, each identified with specific markers ( Table 1) [71, 72] .

One mechanism by which lung epithelia begin to mature is based on chemokine secretions from the surrounding mesenchyme and the developing heart field which are well reviewed here [73] . Key players including fibroblast growth factors (FGFs) [64, [74] [75] [76] [77] [78] [79] [80] , WNTs [81] [82] [83] [84] , and bone morphogenetic proteins (BMPs) [85] [86] [87] [88] [89] [90] are known to induce the differentiation of early lung progenitors in a controlled manner. For example, in mouse, it has been found that FGF10 plays a role in bud outgrowth [77] and drives lung progenitors towards a distal fate [78, 79] through canonical WNT signaling [64, 81, 91] . Proximal epithelia develop because they are located further away from distally located FGF reservoirs in the mesenchyme, in a mechanism that appears dependent on concentration gradients [64] . BMP4 plays a key role in lung bud formation from foregut endoderm and establishment of both dorsoventral (back to front) and proximodistal (top to bottom) patterning in the nascent lung [88] . BMP4 is also present at high levels in distal bud tips and epithelia including AEC II cells [88, 89] , however, its inhibition promotes a proximal fate and, along with BMP2 inhibition, ciliated cell development [87, 88, 90] .

J o u r n a l P r e -p r o o f

While the cell fate of early proximal and distal lineages is directed through chemical signals, the lung epithelium itself undergoes marked changes in architecture, a process known as branching morphogenesis [79, 92] . From the simple tube of the anterior foregut endoderm to the complex tubular structure of the adult, a highly stereotyped mechanism of branching morphogenesis facilitates the outgrowth, division, placement, and structure of lung airway [42] .

Branching morphogenesis of the lung is driven by three simple and iteratively used processes:

domain branching, planar and orthogonal bifurcation [93] . The first form of branching is domain branching: along a primary branch, buds form in a linear and sequential fashion, from proximal to distal. The next form of branching is planar bifurcation, in which the tip of the forming tube bifurcates to create two new tips, which subsequently elongate and bifurcate again, creating four tips. The last process of branching is known as orthogonal bifurcation. In this process, the initial planar bifurcation is followed by a rotation around the planar axis which creates two new tips through bifurcation. A critical gene in this process, Sprouty, has been found to attenuate Erk1/2 signaling, thereby altering the orientation of cell division and future tube elongation [94] . Other critical genes and regulatory networks associated with FGF signaling also contribute to controlling the periodicity of the branched network [95] . Although elements such as domain specification, bifurcation, rotation and branch generation remain largely undetermined [93, 96] , new technologies involving high-resolution live imaging, tension sensing, and force-mapping are opening paths to further explore and explain the branching morphogenesis phenomenon [97] .

The early structure of the lung gives rise to a striking architectural separation of future SOX2 + proximal lineages and SOX9 + distal lineages, at least in mice [98] . The diameter of tube generated during branching morphogenesis in the pseudoglandular and canalicular stages has a small degree of variance within each stage as measured from electron micrograph sources of fetal human tissue [99] . This suggests that the branching program is rigorous in its control of lung structure and that tubes themselves may have instructive potential on the developing epithelia. Once the basic organ structure has formed, the lung continues to be exposed to mechanical cues as it continues to mature. In several cases, these cues have been shown to be essential for correct organ function. In utero, the fetal lung is a secretory organ that only converts to an absorptive one, to prepare for breathing after birth, through a change in the activity of J o u r n a l P r e -p r o o f chloride and sodium channels late in development. Fetal lung secretions result in a static fluid pressure of around 2.5 cmH 2 O in the developing terminal sacs of the fetus, which propels branching morphogenesis outwards into the developing thoracic cavity [100, 101] . Lack of amniotic fluid in the developing lung alters the expression of distal epithelial markers and consequently results in the creation of smaller than normal lungs (pulmonary hypoplasia) [102] , highlighting the importance of this mechanical pressure during lung development. In addition, cyclic strain is generated from fetal breathing movements (FBM) in utero that prime the airway for use after birth. FBM are detectable from the tenth week of pregnancy and begin as infrequent and erratic activity with long quiescent periods. As development continues, these quiescent periods decrease and sustained periods of fetal breathing occur. These breathing movements vary with the fetal sleep cycle and can be chemically tuned [57] , and alter the volume of terminal sacs by around 5% [100, 103] , again highlighting the importance of mechanical signals influencing lung development. Finally, a novel FGF10/FGFR2-dependent tensional mechanism has been shown by which distal epithelial cells in the lung accumulate motor proteins at the apex of the cell, thereby becoming resistant to compression from increasing fluid pressure within the tube lumen. Cells under this tension are more likely to become AEC II cells, while those under compression become AEC I cells [102] . Interestingly, while the above examples highlight the importance of specific mechanical signals in the growth, development, and differentiation of the lung, PSC directed differentiation protocols of the lung are primarily based on mimicking the sequential chemical changes that occur during lung development. 26, 62, 63] , reveals that SOX2 + SOX9 + progenitors are common in the developing lung buds and that branch tips of the pseudoglandular staged lung give rise to both proximal and distal epithelia [63] . Moreover, specific protein markers have been found to differ in both timing and location of expression between human and mouse models: pro-SPC in mouse is expressed early and throughout the developing mouse epithelium [117, 118] , while in human, pro-SPC is rarely detected early in development and is only robustly found later in distal epithelia [63] . These examples highlight that, while there are similarities, development and patterning of mouse and human lungs is different, and these differences require human models to be fully appreciated.

Human PSC protocols have generally followed the same differentiation chronology as that of mouse directed differentiation, wherein definitive endoderm, anterior foregut endoderm, progenitors. In all cases, these lung progenitors are then either sorted or directly guided towards proximal or distal progeny in 2D or 3D culture systems. Ideally, products of directed differentiation protocols should mimic the cell proportions present in human airways and lungs (Table 1) , however current protocols have not progressed that far. While these protocols continue to be refined, the percentage of select cell populations generated from these protocols have been summarized in Table 2 .

Protocols to create proximal lung epithelia have focused on the production of the four major cells types present: ciliated, goblet, club, and basal cells (see Table 1 for a summary of markers for each cell type). Motivation for creating proximal epithelia in the field has primarily been to develop patient-specific cystic fibrosis (CF) models [11, 24, 27] and/or to produce epithelia with multi-ciliated cell populations for protocol validation [30, 33] . A shift towards human PSC-derived CF models has been critical as mouse models do not accurately represent J o u r n a l P r e -p r o o f Journal Pre-proof CF disease progression and phenotypes seen in humans [128] [129] [130] . As such, the first evidence of human PSC proximalization using CF patient-derived PSCs was shown by Mou non-lung hepatic cells (SERPINA1 + , SOX9 + , APOA2 + , AFP + ) [27] . Multi-ciliated cells were only observed within spheroids when NOTCH signaling was inhibited, at the expense of downregulated SCGB1A1 expression, or when spheroid cells were further cultured in ALI conditions. Other mature epithelial populations including club and goblet cells were not assessed post ALI culture. It is unclear whether 2D or 3D culture systems resulted in more representative proximal populations, although it is worth noting that the 3D spheroids could be manipulated to produce a variety of proximal epithelia ranging from progenitor to differentiated populations.

The most recent approach, described by de 

Overall, the aforementioned studies differed drastically from each other with regards to the timing and chemical modulation of each phase of differentiation towards proximal epithelia, and consequently produced variable results. While it is evident that 3D culture augments maturation, no protocol to date has been able to efficiently produce all functional epithelial populations present in the airway in proportions representative of those in vivo. Furthermore, these studies have not thoroughly elucidated the mechanisms of proximal patterning. Barring the application of FGF18 [11] (known to enhance proximal programming [133] ), protocols have adopted growth factors based on trial and error without understanding why, for example, FGF10

signaling (which is known to favor distal lung development) promotes production of proximal progenitors [11, 27, 30, 33, 37, 119] . As such, the quest for obtaining mature airway progenitors, such as NGFR + cells, comes at the cost of elongated protocol lengths, heterogenous maturation levels of resulting populations, and missed opportunities for understanding why these populations do not result in a histologically appropriate epithelium [29] .

It is apparent that the timing of signaling molecule delivery as well as the competence of subjected cell populations to respond to a given signaling molecule are of extreme importance.

The spatiotemporal dynamics of cell signaling are non-linear, are more complex in vivo, and are not fully appreciated in the latter stages of current directed differentiation protocols. This may explain the incongruence amongst different protocols, primarily those assessing the effects of GSK3β and WNT signaling [27, 29, 30] , all of which targeted populations at non-comparable protocol stages. Therefore, a deeper analysis is required to appropriately explain and mimic these dynamics in vitro. Furthermore, recreating these spatiotemporal signaling patterns during directed differentiation protocols may potentially require repurposing molecular delivery tools from other fields such as drug delivery and tissue engineering [134] [135] [136] .

Interestingly, most existing protocols have been skewed towards generating multi-ciliated cells at the expense of goblet and club cells by subjecting airway progenitors to NOTCH inhibition, which is known to decrease goblet cell populations [137, 138] . Goblet cells, in J o u r n a l P r e -p r o o f addition to club cells, have recently been discovered as a source for generating multi-ciliated cells in primary airway epithelia [139] . Club cells play a key role in epithelial injury, wherein they de-differentiate into basal cells in the absence of basal cells such that they can give rise to ciliated and club cell populations to repair a denuded epithelium [140] . Therefore, in the future it will be critical to identify protocols to create PSC-derived cultures containing these cell types,

and not just multi-ciliated cells, in order to fully capture the dynamics of airway injury and repair for drug screening. Overall, based on current progress, the Konishi et al. and McCauley et al.

protocols are considered the most relevant for generating functional airway epithelia.

The alveolar space in the distal lung is comprised of two epithelial cell types: AEC I and AEC II (see Table 1 for specific markers of each cell type Meanwhile, progenitor-like SOX9 + SPC + and SOX9 + HOPX + clusters were prominently present with minimal mature SPC + (5%) and HOPX + (4%) populations. Further refinement of this protocol bifurcated proximal "human lung" and distal "bud tip progenitor" organoid development by culturing foregut spheroids in FGF10 with 1% serum or FGF7, CHIR, and RA in serum-free media, respectively [121] . After 65 days of culture, the "human lung" organoids expressed P63, FOXJ1, and mesenchymal markers with no sign of mature epithelial features; some SPC and HOPX staining was also observed. Only after an 8 week-long in vivo implantation did mature ciliated AcTUB + cells appear. "Bud tip progenitor" organoids also contained heterogenous MUC5AC + , HOPX + , SPB + , and SPC + cells after 120 days. However, when seeded into naphthalene-injured mouse airways, they gave rise to AcTUB + and MUC5AC + cells. In general, this protocol diverged to produced lung organoids with heterogeneous populations of either predominantly proximal or distal epithelia, which required prolonged culture or in vivo implantation for maturation (limited in this case). A key aspect of the "human lung" organoids was their inclusion of a mesenchymal population to study epithelialmesenchymal crosstalk during lung development. implantation to promote maturation (incomplete in this case).

All described directed differentiation protocols for distal epithelia utilized a 3D culture approach in some format, however only those that established lung specification in 2D culture

prior to a 3D transition demonstrated promising results. These protocols do not completely depend on spontaneous organoid assembly, are highly responsive to fine tuning with morphogens, and can therefore provide better insight into the cellular responses in lung development to generate therapeutic strategies accordingly.

Although the "human lung" organoid and "lung bud progenitor" organoid-based protocols may be useful for studying complex cellular interactions during disease progression or Furthermore, the employment of NOTCH inhibition during the "preconditioning" phase may have played a role in promoting both AEC I and AEC II populations, and therefore the aforementioned signaling pathways need to be collectively assessed. Inspiration can be sought from a recent study which described a computational modeling approach, based on single cell RNA sequencing, that predicted the optimal time point for CHIR withdrawal for maintaining a NKX2.1 + SPC + lung fate; its findings were further supported by empirical studies [35] .

Employment of such techniques will prove essential for understanding fate choice and developing customized target lung populations.

The study of airway and lung diseases is limited by animal models as they do not recapitulate human disease phenotypes and progression adequately. For example, existing mouse J o u r n a l P r e -p r o o f models of cystic fibrosis (CF) vary greatly in their ability to represent relevant organ pathologies and are deficient in developing spontaneous lung disease observed in humans [141] [142] [143] .

Similarly, pulmonary fibrosis is most commonly studied in the bleomycin-induced lung fibrosis mouse model, which results in faster disease progression, eventual resolution of disease phenotype over time, and is obscured by the wide range of bleomycin doses administered for induction of injury [144] . Studies exploiting PSC-derived lung culture models to explore the effect of drugs in human lung cells are therefore beginning to emerge.

Culture models of proximal airway epithelia have been applied for drug discovery primarily in the context of CF. CF is an autosomal recessive genetic disorder caused by mutations in the epithelial chloride channel gene, CFTR, which consequently leads to accumulation of excess mucous and compromised mucociliary clearance [145] , affecting multiple organs. CF phenotypes have been studied widely in primary or PSC-derived intestinal [146] [147] [148] , rectal [149] [150] [151] , pancreatic [152] , and airway models [11, 27, 33, 153, 154] .

In the context of airway models, ALI culture has been the gold-standard for studying primary airway epithelia derived from CF patients. In concordance with this method, Wong et al. and developed an IL-11 antibody which reversed late-stage lung fibrosis by significantly decreasing ECM deposition in an animal model [162] . Evaluation of this IL-11 antibody using J o u r n a l P r e -p r o o f PSC-derived lung organoid models can provide better insight into their applicability for human disease.

Distal lung organoids have also been applied to model respiratory viral infections caused by respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV3) [163, 164] , as well as the measles virus (MeV). Chen and colleagues infected LBOs with RSV, resulting in characteristic luminal shedding of epithelia [26] , which leads to small airway obstruction and consequent bronchiolitis in clinical settings [165] . Interestingly, Sach et al. demonstrated that prior incubation with palivizumab (an antibody that prevents RSV from fusing with cells)

prevented RVS from replicating in primary airway organoids [154] , which would be valuable to assess in PSC-derived lung or airway organoids. Meanwhile, HPIV3 infected AEC II in LBOs and temporally reached peak infection similar to that in primary alveolar epithelia [164] , confirming clinical data. HPIV3 infection did not result in either epithelial shedding or syncytium formation in the LBOs as did RSV [26] and MeV [164] infections, respectively, again confirming clinical phenotypes. This showed the ability of lung organoid models to not only demonstrate virus-specific infection, but also to recapitulate phenotypes observed in the clinic.

Another condition modeled by alveolar or lung organoids is SPB deficiency, a lethal neonatal autosomal recessive disease which necessitates lung transplantation for patient survival. 

While there has been great progress in the establishment and maturation of lung epithelia from PSC populations, a number of limitations have emerged that will require optimization and augmentation of current protocols to create better developmental and disease models, and specific cell populations:

a. Lack of control over which populations are produced -understanding or recapitulation of signaling pathways beyond proximodistal patterning is currently limited, as the ratio of AEC II versus AEC I cells; or club cells versus goblet cells cannot be reliably predicted.

Furthermore, while development of reporter lines and identification of surface markers for sorting have [32, 34] helped the advancement of distal lung protocols (for AEC II cells specifically) in the last few years [28, 36] , such techniques are limited in current proximal airway protocols [168] . f. Minimal recapitulation of physiological conditions -current alveolar organoids are beginning to represent relevant cell types, however, they are embedded in Matrigel and grown in submerged culture. They, therefore, fail to provide an ALI environment, which is critical to in vivo functionality. As in proximal protocols, cell products often need to be dissociated and regrown on transwells, which allow ALI culture by exposing cells to media basally and air apically, for further assessment.

g. High cost -associated with the growth factor and small molecules required for chemically directed stem cell differentiation, and the expertise required to reliably create lung epithelia with these protocols. The use of commercially available 2D based endoderm differentiation kits have greatly decreased the level of expertise required to achieve this early stage of differentiation.

Based on current advances, we have made recommendations for directed differentiation protocols that generate proximal and distal epithelia in Figure 2 . 

The timing of chemical signals present during lung development has been well mimicked in current differentiation protocols. The lung, however, develops in response to chemical signals within a highly dynamic mechanical environment of cyclic strain, pressure and a complex branching tubular architecture [169] . Indeed, it is well established that mechanical cues can impact progenitor cell fate [102, [170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] and emerging evidence suggests that the mechanical environment can be manipulated to produce predictable fate choices in stem and progenitor cells [181] [182] [183] [184] . For example, the importance of biophysical manipulation associated with tissue has been exploited only to a limited extent to augment and guide directed differentiation protocols to address some of the above limitations [170, [185] [186] [187] . Organoid cultures, for instance, allow self-assembly of tissue-like structures and enable further maturation of proximal and distal epithelia [12, 26, 28, 119] . In this section of the review, we highlight tools from tissue engineering that have been used to manipulate tissue structure and the resulting biophysical signals experienced during stem cell differentiation in both 2D and 3D (Figure 3 and Table 3 ).

Furthermore, we explore the opportunity to utilize such tools to engineer mechanical signaling as a strategy to augment and refine existing chemical differentiation protocols. Note, we do not consider the use of simply culturing differentiating cells on substrates or in 3D hydrogels with variable mechanical stiffness but point the reader to excellent reviews on this subject [188, 189] .

Micropatterning of the culture surface is one strategy that has been used to manipulate the physical organization of 2D stem cell colonies and the resulting mechanical environment individual cells experience within the cell sheet. Micropatterning entails deposition of J o u r n a l P r e -p r o o f extracellular matrix (ECM) protein islands with highly specific shapes and sizes on non-adhesive surfaces, via micro-contact printing (µCP) [190] [191] [192] [193] [194] or soft lithography [195] [196] [197] . Individual cells or cell populations are thereby restricted to the area of the surface where the adhesive protein islands are present. The shape of the island, therefore, geometrically constrains the shape of single and groups of cells in 2D, which determines the pattern of adhesive attachments between the cells and the underlying surface, and hence the mechanical state of the cells [198] .

Chen and colleagues presented early evidence that geometric constraint affects cell fate by demonstrating that cell growth and apoptosis are directly related to ECM pattern size through its control of cell spreading [194, 199] . Not only is the size of the ECM pattern important, but also the shape it holds, specifically in relation to its aspect ratio and subcellular curvature. As shown by Kilian et al., despite the presence of equipotential differentiation signals, osteogenic differentiation of heterogeneous mesenchymal stem cells (MSCs) was promoted by increasing the ECM pattern aspect ratio at the single cell level [181] . Further, in a pentagon-shaped design, the curvature of the lines connecting vertices was varied from convex to concave and was shown to guide cell differentiation choice from adipogenic to osteogenic, respectively, by manipulating subcellular myosin II polarization, tension, and integrin localization. Evidently, such micropatterned islands not only exert control over the growth and survival of cells, but also enable manipulation of cell differentiation through changes in intracellular tension. For example, by probing tension at the cell-boundary interface through confined 2D geometries, Lee et al.

found that patterned melanoma cancer cells occupying larger arc angles, or smaller magnitudes of curvature, expressed higher cancer stem cell markers [195] . Furthermore, these markers were preferentially found at the edge of the micropattern, a consequence of perimeter tension acting through the p38-MAP kinase pathway. Interestingly, once removed from the defined geometric environment, the cells lost their activated cancer phenotype. These tools could provide an excellent platform for subtly manipulating self-organization of PSC populations to understand and influence their differentiation.

While not in the context of augmenting directed differentiation specifically, the use of micropatterning has been applied to explore pluripotency and fate choice during early development. Based on a previous finding that human PSC differentiation is dependent on colony size [200] , Nazareth et al. developed a high throughput µCP platform, with optimized J o u r n a l P r e -p r o o f colony size, and probed early cell fates (pluripotent, neuroectoderm, primitive streak, and extraembryonic) in response to different media conditions and developmental factors [190] . Such micropatterned surfaces were also used by Warmflash and colleagues to investigate embryonic germ layer patterning of human PSCs [201] . They found that BMP4 treatment results in spatially segregated regions that delineate ectoderm, primitive streak and trophoblast-like tissues within the patterned colonies. This pattern was shown to be mediated by the colony edge, as opposed to colony size, with BMP4 signalling progressively being restricted to the edge of the colony.

These findings were further confirmed by Tewary et al., who explained this effect to be caused by the emergence of a phosphorylated SMAD1 gradient. The establishment of this gradient is initially controlled via inhibition by Noggin, followed by a restriction of BMP4 sensitivity to the edge of the colony due to re-localization of BMP receptors throughout the rest of the colony [202] . Such micropatterned platforms have also been used to map fate choices made during mouse [203] and human [204] gastrulation events, and therefore are a powerful tool for elucidating fate choice during lung development. It is not clear however, how the mechanical state of the cells within these micropatterned islands impacts chemical cue secretion, and hence the local gradients of chemical signals that result in patterning of cell fate in these studies.

Another method to manipulate the physical organization and biophysical cues in a differentiating cell sheet is through substrate texture [205] . Cellular behaviours including proliferation, adhesion, and differentiation have been linked to underlying substrate topographical cues [205] [206] [207] [208] [209] [210] . These cues are recognized by cellular protrusions called filopodia and lamellipodia, through integrin receptors and focal adhesions [211] [212] [213] [214] , which in turn dynamically modify their shape and exert protrusive forces [215] [216] [217] [218] [219] . In this section, we will

provide key examples of substrate topographies, as well as grooves, based on their relevance for lung epithelial organization. These topographies can be microfabricated using various techniques, such as etching, photolithography, soft lithography, and stereolithography, that are scalable, precise, and provide high fidelity [220] .

J o u r n a l P r e -p r o o f

Stem cell fate choices have been shown to respond to topographical features [221, 222] .

For example, Viswanathan et al. assessed the ability of different topographies to mimic sinusoidal epidermal undulation to induce in vivo-like biophysical cues. Their screened undulating topography created β1 integrin patterning that is reminiscent of the human dermis and more differentiated cells were found localized to the troughs of their pattern in a highly repeatable fashion. These findings suggest that replicating the physical organization of the dermal microenvironment promotes tissue-level organization and alters the positioning of the epidermal stem cells towards the in vivo state [223] . This group further applied a screening platform called TopoChip [224] , that incorporated features of varying sizes, roundedness, and distribution density, to assess human PSC proliferation and pluripotency in the absence of ECM coatings [225] . Topographies that ranked high in the screen not only supported PSC proliferation, but also allowed maintenance of OCT4 and SOX2-expressing pluripotent colonies.

In conjunction with computational modeling, this platform was able to predict topographical features conducive to maintaining PSC pluripotency, thus demonstrating great promise for exploring how to use tissue organization patterning to control cell fate. Application of this platform for probing keratinocyte differentiation revealed that differentiation is linked to changes in cell morphology, which is influenced by substrate topography, and mediated by Rho kinase activity [226] .

Based on these studies, it is evident that application of biophysical cues alone can impact differentiation. High-throughput technologies like TopoChip could be used in the future to understand and mimic cell fates of proximal or distal lung epithelia. Towards this goal of recapitulating the physiological morphology of distal lung alveoli, we recently developed largersized topographical features, specifically hemispherical cavities, that enabled seeding of multiple cells and further allowed maintenance of primary AEC I and AEC II cells [227] . The ability of this platform to promote PSC differentiation towards these cell types has yet to be explored, however.

Grooved topographical cues specifically, through their ability to modulate cytoskeletal alignment and cellular shape, have also demonstrated great promise in guiding cell fate [228, J o u r n a l P r e -p r o o f 229]. In the context of neural differentiation, Ankam and colleagues generated a multiarchitectural chip (MARC), that incorporated a range of isotropic and anisotropic topographies at both micro and nano scales, to differentiate PSCs towards neural progeny without the use of embryoid bodies. Anisotropic nanoscale grooves (250 nm) promoted neuronal differentiation with cell alignment and elongation, and isotropic pillars enhanced astrocyte differentiation with cellular branching within 7 days of culture. Meanwhile, conventional culture protocols were unable to induce these populations without additional culture steps and/or prolonged culture up to 30 days [230] . Neural differentiation on nanogrooves was attributed to actomyosin contractility via vinculin-associated focal adhesions [231] . MARC further enabled investigation of nuclear morphology and histone methylation [232] , thereby exemplifying that such platforms can allow exploration of the mechanism of biophysical cues translating to DNA modulation during differentiation.

The influence of groove topography on differentiation has been highlighted in other contexts as well. Abagnale et al. developed a micro-grooved chip, incorporating systematic variation of groove widths and ridges, to study MSC differentiation towards adipogenic and osteogenic progeny. While wider ridges led to higher adipogenic differentiation with formation of fat droplets, thinner ridges enhanced osteogenic differentiation with calcium phosphate precipitation [233] . Interestingly, groove width had minimal impact on favouring differentiation towards either lineage. The ridge-mediated differentiation effect was associated with cell morphology and focal adhesion formation wherein wider ridges resulted in rounder cell morphology with many large focal adhesions, as compared to thinner ridges leading to cellular elongation with fewer and smaller sized focal adhesions. Nano-scale groove topography was also shown to alter the spatial conformation of PSC colonies by elongating them, consequently affecting cell fate [234] . This effect was particularly potent at the colony edges and controlled by separate and differential localization of YAP and TAZ during PSC maintenance and differentiation.

In general, grooved topography can provide great insights into PSC fate choice and potentially expedite differentiation protocols. The MARC platform illustrates the importance of combining biophysical and biochemical cues, especially as exposure to topography induced a higher yield of functional and mature progeny within a short time frame as compared to flat J o u r n a l P r e -p r o o f substrates or standard directed differentiation protocols [230, 235] . This is of extreme importance as current directed differentiation protocols for airway and lung epithelia require longer than 60 days of culture to achieve functional cell types [12, 27, 28, 30, 36, 119] . Although the inclusion of topography has not been investigated for promoting lung differentiation, we have explored grooved substrates for aligning airway epithelia during differentiation to achieve coordinated unidirectional ciliary beating [236] . Our unpublished data demonstrates that while primary human basal-derived epithelia lose their alignment on grooved topography over time, epithelia generated from human PSC-derived airway progenitors maintain their alignment throughout differentiation in ALI culture.

While 2D cues have enabled the community to clearly demonstrate the capability of biophysical cues to manipulate cell fate, 2D approaches are limited in their biological applicability as most physiologic physical and mechanical cues occur in 3D environments [237] . [186] . Further, these microchambers were validated for use in developmental drug toxicity screening, through which they were able to represent Thalidomide embryopathy by exhibiting diminished contractility, beating frequency, and size of cardiac chambers.

Geometric constraint was also applied to cerebral organoids by Lancaster and colleagues to minimize variability associated with neural induction efficiency. This entailed addition of a J o u r n a l P r e -p r o o f physical cue in the form of polymer microfibres, around which the organoid self-organized [170] . In conjunction with an established chemical protocol, these microfibres served to pattern developing organoids leading to recognizable neuronal features including a cortical plate, radial units, along with organized radial neuronal migration in a reproducible manner. This addition of a simple physical cue substantially increased the patterning and organization of brain organoids compared to those derived from protocols only relying on biochemical cues.

Another strategy to control cell and tissue geometry in 3D is the use of micromoulding to create defined mechanical microenvironments which in turn alter cell and tissue level organization and differentiation. This approach was first developed by Nelson et al. using 3D collagen moulds to study branching morphogenesis of mammary epithelial cells [238, 239] .

Seeded mammary epithelia conformed to the 3D architectures, forming hollow tubules, and demonstrated predictable branching patterns according to mould geometry and presence of inhibitory morphogens. This technique was further used to understand the mechanism of cellular rearrangement in mammary ducts [240] and exhibit that mechanical stress gradients control the pattern of branching morphogenesis [241] . Inspired by this method and its applicability for studying lung branching morphogenesis, we developed tubular constructs of physiologically relevant diameters to guide self-assembly of lung progenitors [242] . Using this approach, we demonstrated that specification of these bipotent SOX2 + SOX9 + lung progenitors was dependent on geometry, wherein tubes of 100 µm diameter led to a distal SOX9 + fate, while 400 µm diameter tubes remained in a SOX2 + SOX9 + lung progenitor state. The mechanism of this effect was dependent on canonical WNT signalling, and due to differences in cellular tension induced by patterning the progenitor cells into a 3D tube structure.

While the addition of mechanical cues influences fate choice, its role in inducing cell functionality, especially at the organoid level, needs to be elucidated. Currently, there is scant evidence of lung directed differentiation being manipulated in a 3D context [183, 242, 243] .

Beyond our exploration of patterning early lung fates through micromoulding, Dye et al. have recently applied tissue engineering techniques during lung organoid formation. In their case, foregut endoderm spheroids cultured on highly degradable synthetic polymers demonstrated enhanced ability to differentiate into proximal airway epithelia after in vivo implantation [183] .

Evidently, the field of lung directed differentiation is in its nascent stages for using biophysical 

In the future, human PSC-derived lung tissue models have the potential to enable exploration of infection, disease and regeneration mechanisms of action to impact drug discovery and drug development, and further inform patient-specific drug selection. While lung models remain in their infancy, the investment necessary to translate such models into practical use is worthwhile given that they offer a number of key advantages over primary cells or mouse models. Firstly, PSC-derived platforms enable modeling of human disease. Another major advantage of PSC-derived cells, specifically in the context of lung, is the potential to directly associate specific patient genetics and cell phenotypes with clinical conditions, as is underway in the field of lung cancer [244, 245] . This will avoid complications associated with prior exposure of primary cell donors to a plethora of environmental (such as smoking) and pharmaceutical stimuli. Furthermore, establishing models specifically from PSC sources potentially enables generation of the large number of cells and cell types necessary for personalized disease modeling.

A number of challenges exist however, to translate these models into widespread use for drug discovery and development. One major challenge in the field, highlighted in this review, is the standardization of robust cell manufacturing protocols. Lung epithelial models require not only large cell numbers, but also the correct proportion of cell types. Additionally, for a variety of functional read-outs, these cell types must be appropriately spatially organized. Therefore, standardized protocols are needed to both manufacture lung cells and assemble these cells into reproducible and clinically representative "lung tissues" at the scales required for screening.

This will be essential to enable the generation of in vitro lung test tissues with sufficiently low batch-to-batch and within-batch variation for screening with high reproducibility.

Towards this challenge, methods will continue to emerge to control PSC differentiation into the different proximal and distal lung cell types. For example, in this review, we have highlighted the emerging evidence that an opportunity exists to further improve differentiation control and disease modeling by mimicking mechanical cues experienced during development.

Beyond the strategies described in previous sections, this concept could be expanded further in the future to mimic additional aspects of lung development. For example, the developing lung is exposed to variations in oxygenation [246, 247] , which is known to impact cell fate choices [248] [249] [250] , therefore the use of optimized oxygenation levels could be an attractive and easily scalable strategy to further refine directed differentiation culture protocols. The developing lung is also subject to various other physical cues at the organ-scale including 1) pressure from amniotic fluid, which serves to expand the nascent alveolar compartment; 2) fetal breathing movements that provide a stretch-based physical cue which serves as a maturation signal; and 3)

pulsatile flow from the extensive vascular network present throughout the organ. Techniques that mimic these forces to control lung cell fate are emerging. These include the use of shear to produce relatively homogenous populations of AEC I and II [251] , the use of cyclic mechanical stretch [252] , and the use of patterned hydrogels to enable perfusion of lung-type structures [253] . Scaling some of these complex mechanical setups to enable large scale efficient manufacturing, however, could be a challenge.

The challenge of assembling lung cells into reproducible arrays of "lung tissues" is starting to be addressed by emerging high-throughput techniques that seek to purify specific cell populations [254] [255] [256] that could be later mixed in controlled ratios to generate precise tissue compositions. Bioprinting [257] [258] [259] and cellular assembly efforts, including organoid fabrication through DNA programming [260] , could also potentially prove useful to enabling complex tissue assembly in a manner that is adequately scalable and reproducible for screening. Establishing such "ground-truth" benchmarking data sets to validate the ability of in vitro models to distinguish both positive and negative hits will be absolutely critical to establish confidence in lung culture models and to ensure wider spread community adoption and impact. J o u r n a l P r e -p r o o f Table 3 . Highlights of mechanical cues influencing cell fate. *High throughput not shown in the paper but could be easily developed. 

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FGF-10 is a chemotactic factor for distal epithelial buds during lung development

FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium

Fibroblast growth factor interactions in the developing lung

beta-Catenin maintains lung epithelial progenitors after lung specification

Wnt signalling in lung development and diseases

Hyperactive Wnt signaling changes the developmental potential of embryonic lung endoderm

Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung

Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development

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BMP signalling controls the construction of vertebrate mucociliary epithelia

Bmp signaling regulates proximal-distal differentiation of endoderm in mouse lung development

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Mechanical forces direct stem cell behaviour in development and regeneration

Materials as stem cell regulators

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Geometric control of cell life and death

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Soft lithography in biology and biochemistry

Patterning proteins and cells using soft lithography

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Using self-assembled monolayers to pattern ECM proteins and cells on substrates

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A graphene-based platform for induced pluripotent stem cells culture and differentiation

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Investigating filopodia sensing using arrays of defined nano-pits down to 35 nm diameter in size

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Mechanical stretch induces fetal type II cell differentiation via an epidermal growth factor receptor-extracellular-regulated protein kinase signaling pathway

Multivascular networks and functional intravascular topologies within biocompatible hydrogels

Ultrahighthroughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells

High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting

Situ Expansion, Differentiation and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted

3D Bioprinting Pluripotent Stem Cell Derived Neural Tissues Using a Novel Fibrin Bioink Containing Drug Releasing Microspheres

Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels

Programmed synthesis of three-dimensional tissues

A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte

CellNet: network biology applied to stem cell engineering

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Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

Transcriptional regulatory model of fibrosis progression in the human lung

Defined threedimensional microenvironments boost induction of pluripotency

Graphical abstract