Emerging strategies for stem cell lineage commitment in tissue

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Emerging strategies for stem cell lineage commitment in tissue engineering and regenerative medicine Carley Ort, Khalil Dayekh, Malcolm M.Q. Xing, and Kibret Mequanint ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00532 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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ACS Biomaterials Science & Engineering

Emerging strategies for stem cell lineage commitment in tissue engineering and regenerative medicine

Carley Ort1†, Khalil Dayekh1†, Malcolm Xing2, Kibret Mequanint 1,3* 1

2

3

Department of Chemical and Biochemical Engineering, The University of Western Ontario, 1151 Richmond Street, London, N6A 5B9, Canada

Department of Mechanical Engineering, University of Manitoba, 66 Chancellors Circle, Winnipeg, R3T 2N2, Canada

School of Biomedical Engineering, The University of Western Ontario, 1151 Richmond Street, London, N6A 5B9, Canada

†

These authors contributed equally

* To whom all correspondences should be addressed: E-mail: [email protected] Tel: +1 (519) 661-2111 ext. 88573 Fax: + 1(519) 661-3498

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Stem cells have transformed the fields of tissue engineering and regenerative medicine, and their potential to further advance these fields cannot be overstated. The stem cell niche is a dynamic microenvironment that determines cell fate during development and tissue repair following an injury.

Classically, stem cells were studied in isolation of their

microenvironment; however, contemporary research has produced a myriad of evidence that shows the importance of multiple aspects of the stem cell niche in regulating their processes. In the context of tissue engineering and regenerative medicine studies, the niche is an artificial environment provided by culture conditions. In vitro culture conditions may involve co-culturing with other cell types, developing specific biomaterials, and applying relevant forces to promote the desired lineage commitment. Considerable advance has been made over the past few years towards directed stem cell differentiation; however, the unspecific differentiation of stem cells yielding a mixed population of cells has been a challenge. In this review, we provide a systematic review of the emerging strategies used for lineage commitment within the context of tissue engineering and regenerative medicine. These strategies include scaffold pore-size and pore shape gradients, stress relaxation, sonic and electromagnetic effects, and magnetic forces. Finally, we provided insights and perspectives into future directions focusing on signaling pathways activated during lineage commitment using external stimuli.

Keywords: tissue engineering, stem cell niche, microenvironment, stimuli, differentiation

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1. Introduction Tissue engineering and regenerative medicine continue to be active areas of research due to their potential to transform personalized medicine1-2. Engineered tissues have the potential to impact both fundamental science and translational medicine. First, when human cells are either forming sheets or are seeded into 3D matrices and tissues are fabricated, studies translate better to human outcomes than small animal models3. This provides the potential to significantly reduce the use of animal models in scientific studies and drug discoveries, producing species-specific data relevant to the end goal4. Second, the replacement of damaged human tissues or organs with functional engineered tissues addresses both the mechanical and biological properties mismatch between the native tissue and the graft (e.g., stress shielding in bone implants, compliance mismatch in vascular grafts, and immune response). Since engineered tissues solve these problems, the quest for strategies to fabricate functional human tissues has been an area of significant advance and discovery5. Engineered tissues also serve as models to develop medical devices and study physiological and pathological processes. It can be imagined how such models can be used to study regenerative processes, and how therapeutic drugs or other factors affect such processes to initiate treatment modalities even before performing animal studies6. Recapitulating the native microenvironment of cells in these models will be highly desirable to capitalize on their benefits.

Notwithstanding the progress made, challenges remain which prevent the overall functionality of engineered tissue models; one of which being the effective differentiation of stem cells to specific cell types of interest in the engineered tissue model. Stem cells have the potential to differentiate into multiple cell types, thus having an understanding and control over the factors that determine the differentiation outcome become paramount. 3 ACS Paragon Plus Environment


Based on their potency, stem cells are broadly classified as either pluripotent or multipotent; whereas based on their sources, they can be classified as embryonic stem cells, induced pluripotent stem cells, and adult stem cells. The choices on potency and source depend on the intended potential applications of these cells. This review will highlight recent progress made on stem cell lineage commitment in the context of tissue engineering and regeneration and provides future perspectives (Fig. 1).

Figure 1. Applied strategies targeting stem cell niche for directed differentiation and use in tissue engineering and regenerative medicine. A) Co-cultured specialized cells have been shown to direct stem cell differentiation towards a desired phenotype due to the ability of these cells to secrete a variety of factors and by presenting proteins on their surfaces; furthermore, sequential co-culture with cells from different sources have been shown to guide stem cell differentiation to specific lineage. B) Extracellular matrix is a critical element of the stem cell niche which plays a role in both mechanical support and signaling. Decellularized matrices retain a myriad of growth factors which help seeded stem cells differentiate into the tissue from which the matrix was obtained. Moreover, synthetic scaffolds have been useful in studying stem cell differentiation due to their versatile nature. Synthetic scaffolds can be tailored to conduct electric signals, have a variety of moduli, pore sizes, shapes and topographies, in addition to having the ability to be functionalized or loaded with different factors. C) It is now established that stem cells respond to physiological forces; however, these cells can also be coaxed to differentiate to a specific cell type by using unconventional forces such as sonic vibrations, electromagnetic fields, magnetic forces and stress relaxation. D) By targeting one, or a combination of these elements, it is possible to reduce the non-specific differentiation of stem cells leading to desired cell type and by extension to better, more physiologically relevant engineered tissues.


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2. Lineage commitment induced by co-cultures Historically, cells have mostly been studied in isolation of their native microenvironment. However, this does not reflect physiological conditions. In vivo, tissue-specific resident cells play essential roles in the differentiation of stem cells. Adult stem cells interact not only with the extracellular matrix (ECM) but also with the surrounding cells that exist in the same niche. Neighboring cells provide critical biological signals by means of soluble molecule release and cell surface ligand presentation7. While these signals can be presented to stem cells in vitro, such as by immobilization to materials, the large number of different signals and varying times for exposure may not allow for recapitulation of stem cell differentiation. In view of this, the presence of other cells in co-culture with stem cells can be used as a useful tool to guide in vitro stem cell differentiation and lineage commitment718

.

2.1 Lineage commitment by co-culture with endothelial cells. Endothelial cells have been excellent at coaxing stem cells to differentiate towards mature cells since they replace differentiation factors required for osteogenic lineage commitment7,

15-17

. Different stem

cells such as bone marrow stromal cells and femur-derived stem cells are shown to commit to osteogenic lineage in the presence of endothelial cells19-20. Adipose stem cells cocultured with endothelial cells on poly(L-lactic acid) functionalized with collagen type I has been shown to induce osteogenic lineage commitment without the addition of differentiation factors dexamethasone, ascorbic acid, and β-glycerophosphate. In contrast, the same cells cultured without endothelial cells displayed lower levels of osteogenesis, even with the presence of osteogenic differentiation factors15.



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In the above-cited studies, endothelial cell-mediated bone morphogenetic protein-2 (BMP2) signaling appeared to be the dominant mechanism of action

20-21

(Fig 2). During stem

cell differentiation to osteoblastic lineage, several cytokines are involved, and BMP-2 is considered to be the most active cytokine to induce osteogenic lineage commitment22. Vascular endothelial cells secrete BMP-2 in vivo as an inflammatory mediator in the vasculature, which enhances vascular calcification23. This behavior can be capitalized in

vitro to promote osteogenic differentiation24. BMP-2 binds to type I (BMPR-I) and type II (BMPR-II) serine/threonine kinase receptors25. The signal is then transduced to target genes via Smad-dependent (canonical pathway) or the Smad-independent (non-canonical) pathways for transcription of BMP target genes.26 The stem cell origin is also important in lineage commitment by the action of BMP-2 since adipose-derived mesenchymal stem cells (MSCs) committed to osteogenesis27 whereas MSCs obtained from bone marrow or synovial fluid committed to chondrogenesis28-29. The presence of endothelial cells not only induces osteogenic lineage commitment but they also self-assemble into capillary-like structures. Since prevascularization of engineered tissues is a desired design goal, these cells can provide a dual purpose30. Endothelial cells do not, however, exclusively lead to osteogenic commitment. They can also assist in differentiating stem cells down other pathways, such as MSCs into pericytes and smooth muscle cells31. If a non-osteogenic lineage is the desired goal, competing BMP-2-induced osteogenic lineage commitment could be problematic, leading to partial differentiation and potential calcification.


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Fig 2. Schematic representation of BMP signal transduction. Upon BMP ligand binding to Type II BMP receptors, Type I BMP receptors are cross-phosphorylated, recruiting RSmads (Smads 1/5/8) to the intracellular domain of the Type I receptor and initiating signal transduction via phosphorylation. Activated R-Smads then form a heteromeric complex with Co-Smads (Smad 4) before translocating to the nucleus to regulate gene expression. Inhibitory Smads (Smads 6/7) reside in the nucleus, migrate to the cytoplasm and negatively regulate BMP signaling by inhibiting signal transduction at several points along the pathway. The Type I and Type II receptors thought to be involved in BMP-9 signal transduction are highlighted. Reproduced with permission from ref 21. Copyright 2013 Scientific Research.

2.2 Lineage commitment by co-culture with non-endothelial cells. Instead of starting the culture with two cell types, sequential co-culture of stem cells with two other cell types has also been studied14. For instance, initial differentiation of embryonic stem cells towards an endoderm lineage was accomplished by first co-culturing stem cells with primary hepatocytes. Since hepatocytes are of endoderm lineage, it is not surprising that they are effective in establishing endoderm commitment. To achieve maturation of these endodermic cells into beta cells, they were subsequently co-cultured with endothelial cells. The resulting cells not only displayed upregulated Insulin1, IAPP, and Glut2, but the cells also secreted considerable amounts of insulin compared with other approaches14. Beta cells 7 ACS Paragon Plus Environment


rely on endothelial cells for basement membrane production, as these cells do not produce basement membrane independently, thus underscoring the importance of endothelial cells in pancreatic tissue development. Endothelial cells are thought to provide important signals to aid in cell differentiation because blood vessel integration into maturing tissue is a key regulator of organogenesis14,

16

. Mimicking organogenesis in the signals that stem cells

receive from other surrounding cells is, therefore, an important contributor to cellular differentiation.

Another successful differentiation strategy is co-culture of stem cells with mature cells of the same or a similar lineage of interest12-13. MSCs co-cultured with keratinocytes differentiated to epithelium13 while co-culturing them with chondrocytes resulted in chondrogenesis12. In the latter case, cell differentiation was measured by an increase in collagen type II and SOX9 mRNA expression, and osteogenesis was ruled as unlikely since osteogenic markers type I collagen and RUNX2 decreased in co-culture conditions12. Moreover, MSC markers CD44 and CD166 were found to decrease from 98.6% to 3.54% and from 99.3% to 9.20% respectively with the highest amount of chondrocytes in coculture. These studies suggest that providing stem cells with specific biochemical cues present in a particular lineage can be a useful tool for in vitro lineage commitment.

Other studies achieved differentiation of stem cells through co-culture with cell types from unrelated anatomy and different lineages8-11. Given this, co-culture of cortical neural stem cells with Sertoli cells of the testes enhanced the differentiation of neural stem cells into mature neurons8. Neural stem cells cultured with Sertoli cells showed more prominent axon and dendrite development, as compared to stem cells cultured with differentiation media alone. They also stained positively for β-tubulin III, which is a neural protein marker8. Other studies have also demonstrated the beneficial growth and survival effects of Sertoli 8 ACS Paragon Plus Environment

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cells on neural stem cells, mainly due to the biochemical signals and proteins that Sertoli cells secrete, such as neuroleukin and glial cell-derived neurotrophic factor (GDNF)10-11. Skeletal muscle differentiation can also be accomplished by co-culture of precursor cells with neurons. At the neuromuscular junction, neurons and myocytes interact readily and neuronal release of several factors has shown to positively influence muscle differentiation and behavior. First, the release of acetylcholine by neurons causes muscle maturation9, 32. Second, the release of a ciliary neurotrophic factor from neurons causes differentiation of myotubes. Third, the proteoglycan agrin, released from motor neurons during the development of neuromuscular junctions, increases contraction of tissue-engineered skeletal muscle9. To increase the success of skeletal muscle differentiation from myoblasts (myocyte precursor cells) these precursors can be encapsulated with neurons in GelMA (methacrylated gelatin type A)9. An increased number and longer myotubes, and better alignment of these myotubes were seen, implying improved functionality of these differentiated cells following co-culture with neurons9. Differentiation markers such as Myf-5, myogenin, α-actinin, and sarcomeric actinin were also upregulated, supporting muscle maturation9.

2.3 Lineage commitment by co-culture and hypoxia. Hypoxic microenvironments might induce stem cell communication with other types of cells. For example, it has been found that under hypoxic conditions, endothelial cells secrete a chemokine stromal cell-derived factor (SDF-1) that binds to a receptor (CXCR4) on the surface of circulating endothelial progenitor cells and recruits them to the site of ischemia, leading to neovascularization33-34. A 3D multi-culture model created to study the interaction between MSCs, osteoblasts, and adipocytes under hyperglycemic conditions revealed that MSCs exhibited poor viability and clonogenicity when cultured alone or with osteoblasts in hyperglycemic conditions; however, when co-cultured with adipocytes, hyperglycemic conditions did not affect MSCs 9 ACS Paragon Plus Environment


viability or clonogenicity indicating a protective communication imparted by adipocytes35. Such conditions can be employed in vascular tissue engineering to improve revascularization by recruiting progenitor cells to ischemic tissue in diseases such as myocardial infarctions and strokes or to maintain the viability of stem cells in the case of diabetes. 3. Lineage commitment induced by scaffold microenvironment 3.1 Lineage commitment on decellularized scaffolds. The scaffold microenvironment plays a significant role in the differentiation of stem cells. Naturally occurring, synthetic, or hybrid scaffolds could be employed for stem cell differentiation. It can be difficult to determine the role that each component of the scaffold microenvironment plays on stem cell differentiation. The reason is that most studies also contain biochemical differentiating factors which challenge proper data interpretation36,37. The role of retained growth factors in decellularized scaffolds is more impactful for lineage commitment than the scaffold material itself. When the scaffolds are fabricated by decellularization of native ECM, it is likely that the combination of ECM proteins with residual growth factors may influence cell differentiation and lineage commitment regardless of the scaffold topography or chemistry. In fact, analysis of several critical growth factors demonstrated significant retention following decellularization of kidney38, lung39, and dermal tissues40. While the range of growth factors identified varies greatly with the decellularized tissue type, angiogenesis-related (e.g., TGFβ and VEGF families) and tissue homeostasis cytokines (e.g., glucose homeostasis in kidney tissue) were dominant. Notwithstanding this confounding (due to the presence of multiple growth factors), several lines of evidence suggest the strong suitability of decellularized tissues for differentiating stem cells. Human bone marrow mesenchymal stromal cells (hBMSCs) on collagen types I and II sponges differentiated towards a chondrocyte lineage41. A combination of collagen I, collagen II and 10 ACS Paragon Plus Environment

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either heparan sulfate or chondroitin sulfate was found to be ideal for the commitment of seeded cells into a cartilage-like phenotype, as seen by proteoglycan and collagen secretion, as well as Sox9 and Col2 expression41. Additionally, decellularized spleen matrix has been used to differentiate rat MSCs into cells with similar function, gene expression and protein expression as hepatocytes42. Decellularized skin matrix has also been used as scaffolds for human umbilical cord perivascular cells (a type of MSC) for rapid wound healing in a rat model of diabetic wounds43. Although the role of retained growth factors in decellularized ECM cannot be ruled out, human embryonic stem cells (hESCs) seeded on decellularized kidney scaffolds showed renal lineage commitment without cytokine or growth factor stimulation44. The above studies show lineage commitment toward target tissues when the scaffold is prepared from same tissue (e.g., cardiac differentiation of stem cells on decellularized cardiac tissues)45.

Given the retention of multiple growth factors in

decellularized tissues, further studies will be needed to identify specific factors that are responsible for the differentiation of seeded cells on these matrices.

3.2 Lineage commitment on synthetic scaffolds (Figure 3). Although biological materials have shown success in differentiating stem cells41-42, 46-48 most likely due to retained growth factors38-40, synthetic materials have the advantage of being tuned to different moduli, topographies, pore sizes and shapes, protein presentation, and soluble molecule release. Synthetic materials may also be functionalized readily allowing cells to recognize, bind, and differentiate on the surface49-50. In one such study demonstrating the effectiveness of surface functionalization, a poly(ethylene glycol) hydrogel was functionalized with fibrinogen bound to its surface49. Human pluripotent stem cells were differentiated into contractile heart tissue following encapsulation in this functionalized polymer49. These cells displayed self-alignment of differentiated cardiomyocytes and the tissue modeled physiological heart development. A scaffold composed of tricalcium phosphate-collagen11 ACS Paragon Plus Environment


hyaluronan promoted the differentiation of hMSCs down an osteogenic lineage without the addition of other biochemical factors51. The addition of europium, a rare earth element, to mesoporous bioactive glass for bone tissue engineering leads to the increased expression of ALP, COL1, and Runx2 in bone marrow stromal cells; all osteogenic markers52. These studies collectively show that selection of materials that mimic the matrix chemistry/composition of the stem cell niche is a logical approach. In the next sections, only recent advances are discussed.

3.2.1 Lineage commitment on graphene-based foams and microfibers. Graphene-based scaffolds have recently received much attention as a neural scaffold material due to their biocompatible properties, electrically conductive properties, and ability to enhance neural stem cell differentiation through these properties53-58. Since there are several studies dealing with the role of graphene-based biomaterials in stem cell differentiation under 2D culture conditions 56, 59 and is recently reviewed 60-61, we focus on summarizing 3D culture studies that are more relevant in the context of tissue engineering.

In one of the first studies documented62, graphene foams induced spontaneous osteogenic differentiation of hMSCs without the need for differentiation media. This was likely a cellular response to the high cytoskeletal tension caused by a stiff graphene foam. While neural stem cells (NSC) cultured on graphene foams led to neuronal lineage commitment63, hMSCs did not undergo similar lineage commitment62. Under physiological conditions, neurons are exposed to a large number of action potentials and it seems logical that the presence of electric signals in the MSCs niche specifies their differentiation to a neuronal lineage. In view of this, pulsed electrical stimulation of 3D foams composed of reduced graphene oxide (rGO) microfibers directed neural differentiation of MSCs64 which was not possible in unstimulated graphene foams. Although neuronal differentiation factors were 12 ACS Paragon Plus Environment

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added to the culture media, the 3D foams had enhanced marker gene expression compared to 2D counterparts in an otherwise identical condition. It seems that the 3D foam presented an added signal to NSCs towards lineage commitment. This is a good example of the impact that the niche has on stem cell differentiation where plasticity for differentiation of MSCs into non-mesodermal cell types is demonstrated. Whether neuronal differentiation on graphene is governed by surface properties (e.g., graphene causing selective protein adsorption from culture media onto its surface) instead of bulk material properties (e.g., stiffness) remains to be elucidated. It is reported that when polycaprolactone fibrous scaffolds were coated with graphene oxide of varying concentration, NSC differentiation into

mature

oligodendrocytes

was

readily

achieved

without

the

addition

of

differentiation factors65. What is interesting about this study is that the graphene coating upregulated differentiation genes; however, to a lesser degree than when graphene was coated on the 3D fibers. It seems reasonable to speculate that protein adsorption alone is not likely to be the cause for this observed difference. A more rational argument is that the third dimension imparted by the fibers in conjunction with the graphene coating may have a dominant effect.

3.2.2 Lineage commitment induced by scaffold modulus and topography. Although scaffold elasticity is one of the widely studied variables regarding stem cell lineage commitment, it mainly involves planar surfaces66 which cannot be extrapolated to 3D scaffolds. Furthermore, the exact role of scaffold elasticity should be studied without differentiation factors added to the system. In a well-defined experimental setup, human bone marrow stromal cells (hBMSCs) encapsulated in poly(ethylene glycol) hydrogels with moduli from 0.2 kPa to 59 kPa underwent osteogenic differentiation in the absence of osteogenic differentiation supplements.67 This lineage commitment occurred despite the considerable changes in modulus (~300 fold increase). Interestingly, MSCs cultured with 13 ACS Paragon Plus Environment


similar modulus ranges on 2D acrylamide gels showed multi-lineage commitments to neurons, muscle cells, and osteoblasts66. Although other subtle differences exist to influence lineage commitment (e.g., hydrogel chemistry, ligands), the spatial effects between 2D and 3D is could be a significant factor. The mechanism of lineage commitment also appeared to be different. On 2D gels with varying moduli, mechanosensing and lineage commitment was dependent on non-muscle myosin II and ROCK signaling whereas in 3D, this mechanism was ruled out and integrin signaling was proposed as a possible mechanism. This signal difference in a seemingly similar system is not surprising since it is known that spacial effects (2D vs. 3D) are significant in cell signaling68-69. In comparison to 2D cultures, well-defined 3D cultures lead to a cell-cell signaling landscape more compatible with physiological conditions70. Unlike hBMSCs, the lineage-specific commitment of NSCs is related to the modulus of silk nanofiber hydrogels in the absence of biochemical factors.71 By annealing the fiber hydrogels using water/methanol mixtures to change the modulus, NSCs on a low modulus (0.57 kPa) differentiated into neurons whereas those cultured on 1.3 kPa modulus fibers differentiated to astrocytes. Human MSCs embedded in gelatin-hydroxyphenylpropionic acid also expressed markers for neuronal commitment in the absence of biochemical factors72.

Topographical features of scaffolds have been known to influence cell differentiation.73 Rather than discussing the well-documented effect of a 3D macrostructure on cell differentiation and behavior74-78, recently emerged novel microstructure modulation is emphasized here. Surface roughness gradient is one parameter that is studied for osteogenic differentiation of hMSCs79. The rationale for studying surface roughness in the context of bone tissue is to mimic the in vivo bone microenvironment. Scaffolds with rough inner surfaces that are modulated topographically with grooves and pits enhanced osteogenesis of hMSCs80-81 and hESCs82 even without soluble inducers. This is supported by an 14 ACS Paragon Plus Environment

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independent study where the most superior osteogenic differentiation of hBMSCs was obtained when cultured on hydroxyapatite discs with an average roughness between 0.77 and 1.09 µm and a mean distance between the peaks of 39.3 to 53.9 µm83. Stem cells cultured on discs of this topography displayed an aligned and elongated organization along the grooves, with F-actin fibers mirroring this organization83. To add to this assertion, human osteoclast precursor cells showed the highest differentiation and cellular function on surfaces of hydroxyapatite with topographical grain diameters of 3.9 µm and average roughness of 0.243 µm84. The resorptive capabilities of the differentiated osteoclast cells and calcium ion concentration were measured from the media of cells cultured on either surface. It was also found that the larger grain diameter and roughness values promoted higher calcium ion concentrations, implying better resorptive capabilities84.

Topography can also be modulated in terms of the surface area in which cells are restricted. When MSCs were restricted to a surface area of 1000 µm2 using fibronectin squares, differentiation to adipose cells was seen85; while a surface area of 10,000 µm2 led to osteoblasts85. This approach suggests that MSCs could be constricted using fibronectin squares of a specific surface area and allowed to differentiate before they are seeded into a 3D scaffold. Furthermore, 3D printed bioinks are becoming popular due to the high control over topology and geometry of the stem cell niche. It has been shown that even the pattern of cell seeding in 3D printed bioinks can affect the differentiation of stem cells. An alternating pattern of human cardiac progenitor cells and MSCs in patches made of porcinesourced bioink were implanted in a rat myocardial infarction model and showed better vascularization and cardiomyocyte differentiation than randomly mixed co-culture patches86. This shows that while co-culture of cells may have an advantage over a single culture of stem cells, a microenvironment of an organized pattern of a co-culture can amplify and accelerate the benefits. 15 ACS Paragon Plus Environment


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3.2.3 Lineage commitment induced by scaffold pore size and pore geometry. Pore size is another scaffold property that can be modulated in order to differentiate cells towards specific lineages 87-89. Collagen-hyaluronic acid (CH) freeze-dried scaffolds having a mean pore size of 300 µm promoted chondrocyte differentiation of hMSCs better than 94 µm mean scaffold pore size as evidenced by Sox9 and COL2 expression87. Not only differentiation but also cartilage ECM production was enhanced on larger pore size scaffolds suggesting that pore size induces chondrogenic lineage commitment in otherwise identical conditions. Similarly, polycaprolactone (PCL) scaffolds fabricated by particulate leaching and having 200-300 µm pore sizes promoted differentiation of hMSCs towards osteogenesis, while 300-450 µm pore sizes supported chondrogenic differentiation88. These studies documented differentiation from a strictly functional level, by looking at calcium mineral deposition for osteogenic differentiation and glycosaminoglycan deposition for chondrogenic differentiation. Instead of fabricating individual scaffolds with different pore sizes, a gradient of pore sizes can also be created on a single scaffold.

Indeed,

poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) scaffolds with gradient pore sizes ranging from 245 µm to 550 µm in the axial direction were fabricated89. On the lower pore size zone, hMSCs began displaying chondrogenic markers such as Sox9 and aggrecan. Additionally, chondrocyte behavior was apparent, as cells produced a higher amount of glycosaminoglycans, leading to denser ECM than scaffolds without pore gradients89. The design is rationalized on the basis that native articular cartilage displays a gradient in ECM composition and organization, and the 300 µm pore size identified to promote chondrogenesis is consistent across the three cited studies. Creating a microenvironment based on pore geometry presents a new strategy to guide lineage commitment90. Taking advantage of additive manufacturing technologies, stem cell instructive scaffolds with different pore geometry can be designed91. Although the 16 ACS Paragon Plus Environment

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mechanism is yet to be elucidated, different scaffold pore geometries are known to influence stem cell fate. Recent studies suggest that rhomboidal pores enhance osteogenic differentiation of hMSCs whereas square and cubic pores supported a better chondrogenic differentiation92-94. Further, scaffolds containing pores of cylindrical geometry increased the expression of early osteogenic markers OLP and APN, while an ordered cubic geometry is more advantageous later in differentiation to assist in calcification and protein expression 92

.

Figure 3. The effect of scaffold physical properties on stem cell behavior. Synthetic scaffolds have the advantage of being tailorable and tunable to the desired use. A) Neural stem cells seeded on 3D graphene foams differentiated into neurons which were responsive to electrical pulse as shown by the increased intracellular calcium (reproduced with permission from ref 63. Copyright 2013 Springer Nature). B) MSCs were found to differentiate to either neural, myogenic or osteogenic lineages based on the elasticity of the substrate they were seeded on as indicated by the type of marker they express (reproduced with permission from ref 66. Copyright 2006 Elsevier). C) The importance of scaffold roughness 17 ACS Paragon Plus Environment


gradient on osteogenic differentiation of human bone-marrow MSCs. MSCs that were seeded on areas of the scaffold that corresponded to a roughness less than 3 µm, and mean spacing between peaks larger than 50 µm (5mm and 10mm) showed higher ALP activity even without osteogenesis inducing media (reproduced with permission from ref 79. Copyright 2015 Elsevier). D) Pore size is yet another important property for synthetic scaffolds to mimic native tissue. Scaffold G had a gradient of pores ranging from 50 µm to 1100 µm, scaffold NG500 is a non-gradient scaffold with 500 µm pores, and NG1100 is also a non-gradient scaffold but with a pore size of 1100 µm. Mesenchymal stromal cells seeded on the G scaffold produced more calcium (blue) and phosphate (green) than the other scaffolds. Moreover, the shape of pores was shown to affect stem cell behavior (reproduced with permission from ref 91. Copyright 2016 Springer Nature). E) qRT-PCR analysis from cells seeded on cubical (light blue) or cylindrical (dark blue) pore scaffolds. While cells on cylindrical pores show higher osteopontin expression, those on cubical pores show a higher expression of adiponectin and type II collagen (reproduced with permission from ref 92. Copyright 2016 Elsevier). 3.2.4 Lineage commitment induced by biomolecule-conjugated scaffolds. Proteins and biologically active molecules can be bound to scaffolds to modulate the differentiation of seeded stem cells95-96. Specific ligands can be attached in one of three ways: (i) direct conjugation to the scaffold, (ii) conjugation via a flexible molecular arm, and (iii) noncovalent adsorption. These three methods were tested for signaling efficacy by immobilizing the cytokine LIF either directly to poly(octadecene-alt-maleic anhydride) (POMA), indirectly to POMA through a PEG spacer arm (where the proximal end of the arm is covalently bound to the scaffold), or noncovalently to extracellular matrix proteins overlaying the POMA surface97. Covalently binding LIF directly to POMA led to the highest ligand density, whereas having LIF bound through PEG7 spacer arm increased hydrophilicity and mobility of the biologically active compound as well as a further distance of action to react with surrounding cells. This, in turn, led to higher antibodyaccessibility of the ligand. Both covalently bound methods resulted in dose-dependent LIF signaling and interestingly, induced LIF signaling to a similar degree97. Noncovalent binding of the ligand to the surrounding extracellular matrix resulted in degradation of LIF over the 72 h study, as opposed to the two covalently bound groups, which maintained bioactivity for the length of the study97. This approach has implications for tissueengineered scaffolds, if sustained exposure to a bioactive molecule is required. 18 ACS Paragon Plus Environment

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Conjugating specific molecules to scaffolds can aid in the differentiation towards a lineage of interest. For osteogenic differentiation, BMP-2 has been effectively utilized as a bioactive molecule98. A polydopamine-assisted immobilization to poly (lactic-co-glycolic acid) (PLGA)-[Asp-polyethylene glycol (PEG)]n copolymer scaffolds led to the expression of osteocalcin and type I collagen − markers specific to bone development96. Viral nanoparticles have also been explored for this purpose of exposing stem cells to an active molecule for differentiation95. Specifically, tobacco mosaic virus was investigated by binding the arginine-glycine-aspartic acid (RGD) sequence to the surface of the viral nanoparticle, via tyrosine residues, using a copper(I) catalyzed azide-alkyne cycloaddition reaction95. The rationale behind using this particular viral nanoparticle comes from its documented ability to increase osteogenesis95, 99-101. This method has implications for tissue engineering, as it could allow the functionalization of scaffolds with tobacco mosaic virus. The advantage of covalently bound biologically active molecules to the surface of the scaffold is the sustained exposure of these ligands to the cells97. 3.2.5 Lineage commitment induced by biomolecule-loaded scaffolds. Bioactive molecule release can be a promising tool for the differentiation of stem cells towards a favorable lineage13, 102-105. Smooth muscle cell differentiation from human amniotic fluid stem cells was successful by providing these cells with PDGF-BB and TGF-β1 in the media102. In vitro culture of these stem cells showed that these two bioactive molecules, when used together, stimulated cells to display a high level of α-smooth muscle actin, desmin, calponin, and smoothelin - all markers of smooth muscle cell differentiation102. Functionally, these cells also contracted, illustrating the effectiveness of these molecules as differentiation signals102. The release of PDGF-BB and TGF-β1 from a scaffold could be employed in vascular tissue engineering, for the achievement of contractility of the tissue construct. If a neural lineage is of interest, it has been found that releasing LY294002 (a 19 ACS Paragon Plus Environment


PI3K/Akt pathway inhibitor) from electrospun PCL-collagen scaffolds promotes the differentiation of stem cells to neuronal cells105. Moreover, from a morphological viewpoint, cells developed projections similar to axons and dendrites105. The release of magnesium ions (Mg2+) from β-tricalcium phosphate scaffolds has also shown to be an effective strategy for the differentiation of MSCs towards osteogenesis, increasing the activity of alkaline phosphatase in cells, as well as upregulating the expression of osteopontin and osteoprotegerin104. When released from a nanofibrous composite scaffold composed of hyaluronan, chondroitin sulfate, and cationic gelatin, differentiation towards an epithelial lineage was observed13. This differentiation was supported by the increased expression of keratin 14, Pan-cytokeratin, and ∆Np63α: dermal protein markers13. Another noteworthy method for controlled delivery of bioactive molecules is from a poly(ester amide) electrospun scaffold, with highly biocompatible degradation products106, which is a promising tactic for cell differentiation, tissue regeneration, repair, or drug delivery107-108. 3.2.6 Lineage commitment induced by stress relaxation (Fig. 4C) Stress relaxation refers to the isothermal and time-dependent stress required to maintain a material at a constant length. Since the length is kept constant, there are no macroscopic changes in the stressed form; thus stress relaxation occurs only on a molecular scale by molecular relaxation due to chain session or due to viscous flow. Many biological tissues exhibit viscoelastic properties with varying relaxation times justifying the need to design scaffold materials with different stress time constants109. While the role of substrate stiffness on stem cell fate is widely studied, the influence of substrate stress relaxation on cell behavior is only emerging110. Polyacrylamide hydrogels with a constant elastic modulus but variable viscous moduli can be prepared by changing the amount of the crosslinking agent111-112. Hydrogels with a high viscous modulus have low relaxation 20 ACS Paragon Plus Environment

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constants and high creep. Myogenic, osteogenic, or chondrogenic differentiation of hMSCs was seen depending on the viscous modulus of the gel, without additional differentiation inducers. A propensity for myogenic differentiation was seen on hydrogels having high viscous modulus. With added differentiation medium for the respective lineages, the viscous modulus was the dominant force for hMSC fate decision. This study elucidates that the scaffold modulus – which is the sum of elastic and viscous – alone is not predictive of how stem cells behave. More recently, alginate hydrogels with varying stress relaxation rates but all with an initial elastic modulus of either 9kPa or 17kPa were prepared by modulating the molecular weight of alginates as well as their crosslinking densities113. Low molecular weight alginate hydrogels increased the stress relaxation rate significantly, which in turn, led to increased osteogenesis of murine MSCs113. Specifically, it was found that for hydrogels of 17kPa and stress relaxation times between 300 and 2300 sec, alkaline phosphatase staining increased as well as mineralization of the matrix and type I collagen secretion 110, 113. Hydrogels with 9kPa initial elastic modulus promoted adipocyte differentiation of murine MSC regardless of the relaxation time (defined as the time required for the stress to drop to 50% of its initial value). However, hydrogels with 17kPa initial elastic modulus promoted osteoblast differentiation in a time-dependent relaxation manner; the fastest relaxation time (1 min) being the most effective one as shown by a mineralized, collagen-1-rich matrix similar to bone. Consistent with this in vitro observation, fast-relaxing hydrogels (relaxation time of 50s) showed significantly more new bone growth in mice models than those that received slow-relaxing, stiffness-matched hydrogels. 4. Lineage commitment induced by non-conventional mechanical signals (Fig. 4). Biochemical signals have always been the major focus to the study of cell physiology; however, accumulating evidence for over a decade has demonstrated that mechanical forces 21 ACS Paragon Plus Environment


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can be just as important to the physiology of cells. The significance of mechanical forces has become increasingly evident in the field of tissue engineering, as these forces regulate vital cellular processes such as proliferation, differentiation, and even apoptosis114-115. Stem cells are affected by and respond to mechanical force signals, greatly influencing their differentiation116. By recapitulating the developmental mechanical forces cells are exposed to, stem cell differentiation towards that particular lineage is influenced. Differentiation of stem cells towards endothelial lineage is responsive to shear forces induced by fluid flow117-118 whereas chondrogenic lineage requires compression forces

119-121

. Since the

effects of physiologically relevant forces on lineage commitment are frequently reviewed116,

118, 122

, we instead focused on unconventional, non-physiological forces for

stem cell differentiation. These forces are sonic vibrations, electromagnetic fields, magnetic forces, and rapid stress relaxation. 4.1 Lineage commitment induced by sonic vibration and electromagnetic fields Vibration involves the back and forth transfer of energy between its potential and kinetic forms. In a damped system, some energy is dissipated in each cycle of vibration and if a steady vibration is to be maintained, this must be replaced from an external source. Sonic vibration stimuli can, therefore, accomplish this sustained stimulus and be used to guide stem cells into a particular lineage such as neural, bone, and cartilage123-127. Both sub-sonic (

Emerging strategies for stem cell lineage commitment in tissue

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