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REVIEW

Engineering Concepts in Stem Cell Research

Karthikeyan Narayanan,* Sachin Mishra, Satnam Singh, Ming Pei, Balazs Gulyas,and Parasuraman Padmanabhan*

The field of regenerative medicine integrates advancements made in stemcells, molecular biology, engineering, and clinical methodologies. Stem cellsserve as a fundamental ingredient for therapeutic application in regenerativemedicine. Apart from stem cells, engineering concepts have equally contrib-uted to the success of stem cell based applications in improving humanhealth. The purpose of various engineering methodologies is to developregenerative and preventive medicine to combat various diseases anddeformities. Explosion of stem cell discoveries and their implementation inclinical setting warrants new engineering concepts and new biomaterials.Biomaterials, microfluidics, and nanotechnology are the major engineeringconcepts used for the implementation of stem cells in regenerative medicine.Many of these engineering technologies target the specific niche of the cellfor better functional capability. Controlling the niche is the key for variousdevelopmental activities leading to organogenesis and tissue homeostasis.Biomimetic understanding not only helped to improve the design of thematrices or scaffolds by incorporating suitable biological and physicalcomponents, but also ultimately aided adoption of designs that helped thesematerials/devices have better function. Adoption of engineering concepts instem cell research improved overall achievement, however, several importantissues such as long-term effects with respect to systems biology needs to beaddressed. Here, in this review the authors will highlight some interestingbreakthroughs in stem cell biology that use engineering methodologies.

1. Introduction

Bioengineering is an interdisciplinary field involving expertisefrom all sciences and engineering with the ultimate goal ofimproving health. Organs form through the assembly ofspecialized cells to perform an assigned function. Materials

Dr. K. Narayanan, Prof. M. PeiStem Cell and Tissue Engineering Laboratory,Department of Orthopaedics and Division of Exercise PhysiologyWest Virginia University,PO Box 9196, One Medical Center Drive,2 Morgantown, WV 26505-9196, USAE-mail: [email protected]

S. Mishra, S. Singh, Prof. B. Gulyas, Dr. P. PadmanabhanLee Kong Chian School of MedicineNanyang Technological University,59 Nanyang Drive, Singapore 636921, SingaporeE-mail: [email protected]

The ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/biot.201700066.

DOI: 10.1002/biot.201700066

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used in regenerative medicine have be-come an essential centerpiece of study; thekey engineering involved in developingthese materials is discussed in this review.The stem cell niche is unique and anydisturbances to that homeostasis will affectthe phenotype of the cells leading to a lossof stemness. Materials and other biologicaland chemical cues have been used todevelop a highly controlled directed differ-entiation. This review comprehensivelydiscusses the various types of engineeringconcepts adopted in regenerative medicine.

2. Synthetic Biomaterials toModulate Stem Cells

Biomaterials are a key component ofregenerative medicine which provide themuch needed microenvironment for theexpansion and improved functionality of thecells they harbor. Biomaterials can bedesigned using biologic or synthetic materi-als. Synthetic materials have several advan-tages over biologically derived materials; forexample, bulk production and reproducibil-ity are some of the key characteristics.Syntheticmaterials can be broadly classifiedinto degradable and non-degradable materi-

als (Figure 1). Degradable materials are preferred for transientsupport in the body; typically, physicians prefer degradablematerials for applications such as cargo delivery or to avoid asecond surgery to remove the implanted material. On the otherhand, in certain situations, physicians prefer non-degradablematerials for reasons that warrant the presence of the material inits original form and composition.

The common chemistry used in degradable materials revolvesaround ester, amide, anhydrides, carbonates, phosphates, orthioester chemical bonds. The table in Figure 1 shows some ofthe chemical bonds and their structure as well as applications ofthese materials in biology. In addition to the chemical structure,other physical properties such as porosity, size, and shape dictatethe degradation rate of these polymers. Degradation of thematerials happens mainly because of water sorption or changesinmechanical properties. Factors such as repeat units, molecularweight, polydispersity, configurational structure, processingmethod, site of implantation, and external physical forces alsocontribute to the kinetics of degradation.

Non-biodegradable materials contain components that resistdegradation by cellular enzymes and microbes. These materials

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Parasuraman Padmanabhan is aDeputy Director, TranslationalNeuroscience in LKC School ofMedicine, NTU, Singapore. Dr.Padmanabhan expertise inmultimodal molecular imaging

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are intended to stay in the host permanently. Since thesematerials need to stay in the body for a long time, their toxicityand bio-inertness are the key characteristics that are essential.Bio-inert materials do not initiate a response or interact withbiological components in the host tissue/body. Initiating aresponse in the host would result in the formation of fibroids andeventually lead to loss of function.[1]

approaches in preclinical area ofresearch. He has a very strongscientific background in cell andmolecular biology, microbial

genetics, probe development and molecular imaging. Hehas more than 25 years of research and teachingexperience gained from several leading universities, suchas Cornell and Stanford University, USA and also fromSBIC, an A*STAR R&D Research Institute, which is one ofthe premier institute in Asean region.

Karthikeyan Narayanan is a post-doctoral fellow at Department ofOrthopaedics, West VirginiaUniversity. Prior to this Dr.Narayanan worked at Institute ofBioengineering and Nanotechnology,Singapore. He has extensiveresearch experience in connectivetissue biology, molecular biology, cellbiology, cell manipulation, and stem

cell research. One of his area of research interest includesunderstanding the niche of stem cells, especiallyextracellular matrix and how it controls stem cell fate.

2.1. Synthetic Biomaterials for Tissue Engineering andRegenerative Medicine

Biodegradable materials are useful in many regenerativeapplications. Poly(lactic-co-glycolic acid) (PLGA) hollow fibermembranes prepared using the wet phase inversion methodwere used in nerve tract guidance conduits.[2] Poly(glycolic acid)(PGA) is a material that has been successfully used in variousapplications including bone,[3] cartilage,[4] tooth,[5] tendon,[6]

intestinal,[7] vaginal,[8] and spinal regeneration.[9]

Non-biodegradablematerials were first used in vascular relatedapplications; they had to be hemocompatible and could not induceblood clots.An interestingobservation is thatno syntheticmaterialis completely bio-inert; however, some materials that are close tobio-inertness include titanium-aluminum vanadium alloy (cur-rently used in hip replacements), diamond, and phosphorylcholine (used in contact lenses). Heart valves with flexiblepolymers have been designed over the years; several syntheticmaterials such as silicone, polytetrafluroethylene (PTFE), andpolyurethanes (PU) have been used. PU has better biocompatibil-ity and bio-inertness when compared to PTFE.[10–12] The design ofthe heart valve primarily involves hemodynamic aspects. Long-term evaluation of heart valves includes thrombosis[13] andcalcificationstudies around thesite of implantation.[14] Toenhancelong-term performance of the implants, especially to improvebiocompatibility and to overcome wear/corrosion, it is a commonpractice to incorporate specific modifications to the surface of thematerial. Surface modifications include physical deposition,[15]

plasma coating,[16] or chemical treatment.[17] Essentially, thesemodifications reduce protein adsorption, cell attachment, matrixproduction, and calcification.

Intraocular lenses (IOLs) for cataract surgery are one of thebest examples where synthetic materials have been usedsuccessfully. The materials and design for the IOLs mustpossess bio-inertness to avoid long-term complications. Earlierversions of polymethylymethacrylate (PMMA) were non-foldableand were treated with heparin to yield better results.[18] Severalother materials, both hydrophobic and hydrophilic in nature,have been tried for IOLs. Hydrophobic materials, such asacrylate based polymers, and hydrophilic materials, such ashydroxyethyl methacrylate (HEMA) have been used for IOLs.The design of IOLs has evolved over decades and has primarilyfocused on the prevention of capsule formation; one suchmodification was the introduction of sharp edges.[19]

2.2. Synthetic Materials for Stem Cell Culture

Both adult and pluripotent stem cells have enormous potentialfor regenerative medicine.[20,21] Expansion of stem cells requires

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the use of specific extracellular matrix proteins (ECM). Many ofthese ECM components were derived from animal resourcesleading to variability, xenogeneic contamination, and immuno-genicity.[22] In order to move stem cell research to the clinicalsetting, synthetic materials that are consistent, cost-effective, andbiologically compatible must be developed. Polymeric biomate-rials have been developed to culture pluripotent stem cells. Forexample, an acrylate-peptide polymeric material was developedand successfully marketed by Corning as Synthemax.[23]

Synthemax enabled self-renewal of human embryonic stemcells (hESCs) and further demonstrated the ability to potentiallydifferentiate toward a cardiac lineage. Villa-Diaz et al. usedsynthetic polymer poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) to culturehESCs.[24] Klim et al. screened various cell adhesive peptidesand reported that peptides that bind to heparin via glycosami-noglycans were effective for long-term culture of hESCs.[25]

Bafman et al. used poly(methyl vinyl ether-alt-maleic anhydride)(PMVE-alt-MA) material to culture hESCs and inducedpluripotent stem cells (iPSCs) for up to five passages.[26] Mostof the studies involved in developing synthetic materials forpluripotent stem cell culture limit themselves to five to tenpassages. The above mentioned materials rely on specific

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Figure 1. Synthetic materials and common chemistry used. A) Use of synthetic materials in different areas. B) Classification of synthetic materials andtheir key features. Unique properties are highlighted in red. C) Common chemistry used in the synthesis of various materials.


peptides which elevate material costs. Our earlier results indicatethat coating polysulfone (PSF) with polymerized 3,4-dihydroxy-l-phenylalanine (DOPA) enabled culturing of pluripotent stemcells.[27] We have demonstrated long-term culture (ten passages)of both hESCs and iPSCs. One disadvantage of this system is theopaqueness of the PSF membrane. In our recent studies,we have developed a simple synthetic material without anypeptide that can be used to expand pluripotent stem cells. Wehave demonstrated the ability to culture pluripotent stem cellsfor more than 23 passages (Patent WO2017095333A1, J. Y. Ying,N. Erathodiyil, K. Narayanan, A. C. A. Wan, (Agency For Science,Technology And Research) Singapore PCT/SG2016/0505872017). The above mentioned synthetic substrates (Table 1) wereused in two-dimensional (2D) culture format; however, recently,several strategies were attempted for growing stem cells in three-dimensional (3D) format. Hydrogels act as excellent materialsfor the self-renewal stem cells. Hyaluronic acid (HA) hydrogelwas developed by Gerecht et al. to propagate hESCs. Ultravioletlight (UV-light) polymerizable methacrylated HA was used toencapsulate hESCs; interestingly, at the end of the cultureperiod, hyaluronidase was used to release cells.[28] We havedeveloped a similar system with HA-tyramine (HA-Tyr) withhorseradish peroxidase (HRP) and hydrogen peroxide (H2O2).This system can be tuned to a specific mechanical stiffnesswhich would additionally augment downstream differentiation

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processes.[29] Although common 2D cell culture substrates havebeen widely used in routine research applications, 2D systemmay not able to supply the required number of cells for clinicalapplications. To obtain one billion cells, it is estimated that 100T75 flasks must be cultured. Stem cell researchers worldwidebelieve that using a 3D culture system would yield the requiredamount of cells for clinical applications. Moreover, 3D cellculture systems provide cell–cell interactions and enhancedifferentiation properties.

2.3. Synthetic Materials-Enabled Stem Cell Research

Stem cells are a key element in tissue engineering andregenerative medicine. The application of stem cells beginswith derivation of specific functional cells that potentially replacea lost function in humans and helps to develop models for drugdiscovery research. Derivation of specialized cells from stemcells is achieved via several differentiation steps which weretranslated from developmental biology. Differentiation of stemcells requires appropriate growth factors and other differentia-tion stimulators. Differentiation stimulators include biochemi-cal signaling molecules and mechanical stiffness that arepresented to stem cells at the right time with the right dose andin the right place. Understanding the native tissue structure,




Table 1. Table shows a list of 2-D synthetic substrates used for pluripotent stem cell culture, cell lines used, properties of karyotyping, methodused for passaging cells, and use of biological entities.

Substrate Cell lines used Karyotyping Passaging method Peptides

Synthemax[30] H1 (10); H7 (10) Data not shown Collagenase Yes

StemAdhere[31] H1 (>60); h9 (>60); iPSC (>60) H9 normal Accutase Yes

Peptide-SAM[25] H1 (6); H7 (6); H9 (19); H13 (14); H14 (17); IMR-90-1 (7) All normal except H14 Manual Yes

PMVE-alt-MA[26] HUES1 (5); HUES9 (5); iPSC (5) HUES1 and HUES9 normal Accutase No

PMEDSAH[24] BG01 (3); H9 (10) Normal Mechanical No

APMAAm[32] H1 (>20); H9 (>20) Normal Collagenase No

15A-30%[33] BG01 (5); WIBR3 (5) Normal Collagenase No

UV/Ozone[34] BG01 (10); WIBR1 (10); WIBR3 (10) Normal with ROCK inhibitor Collagenase No

HG21[35] RH1 (>20); H9 (9) Abnormal Thermal induction No

DOPA/PSF[27] HUES7 (10); BG01 (2);H1 (10); H7 (10); IMR-90-4 (10);iPSC-4 (10); iPSC DF6-9-9T.B (10); hFib2-iPS4 (10)

H1 and IMR-90-4 normal Dispase/Accutase No

Acrylate nanoparticles[36] BG01 (10); HUES7 (10); DF6-99 (10) Normal Dispase/Accutase No

Number of passages tested for each cell lines are listed in bold font within parentheses.


composition, and physical properties enable us to developbiomimetic materials that can stimulate stem cells to undergodifferentiation toward a specific lineage. A wide variety ofsynthetic materials with tunable properties have been used todictate stem cell differentiation. Degradable PGA, poly(lacticacid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) scaffoldshave been used to culture hESCs in a 3D format culture systemand for differentiation. It is favorable to have a system such asscaffolds degrade over a period of time leaving differentiatedcells.[37] Natural polymers such as alginate, chitosan, and gelatinwere used tomake scaffolds of different structure such as fibrousor hydrogel to culture and differentiate stem cells.[38] We havereported a 3D microfiber system to culture and differentiatehESCs.We have used a chitosan/alginate complex to encapsulatehESCs and, following expansion for the required time, hESCswere allowed to differentiate in scaffolds to a neural lineage withover 90% of cells expressing neuronal markers. This method ofexpansion and differentiation potentially generates large-scaleproduction of specific cells for clinical and translationalapplications.[39]

Mechanical stiffness and topography plays a critical role instem cell differentiation. Several research papers suggest therequirement for specific stiffness and topography to obtainhighly functional cells. Mechanical stiffness and nano-topogra-phy are sensed by cells via various integrins which furtherinitiates signaling cascades leading to altered gene expressionprofile. Yim et al. showed that human mesenchymal stem cells(hMSCs) grown on nanograting preferentially differentiatetoward a neural lineage.[40] Nano-topography (patterning),nano-grooves, nanotubes, and nanopits have been utilized tocontrol stem cell fate.[41–43] Biomaterials have been designed toselectively induce or direct the differentiation process towardspecific lineages. Materials with specific mechanical properties,specific ligands, and soluble factors have enabled the differenti-ation process to be more efficient for the differentiation ofneurons,[44,45] cardiovascular tissue,[46] and liver tissue.[47]

Material stiffness is one of the critical factors that regulate

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stem cell fate, adhesion, differentiation, and migration. Manysynthetic materials have tunable properties via cross-linking thedensity of polymers. The effect of material stiffness was studiedusing hMSCs in polyacrylamide (PAAm) hydrogels with varyingstiffness between 1 and 100 kPa.[48] Musah et al. demonstratedthat glycosaminoglycan hydrogel with stiffness �10 kPa wasessential for maintenance of hESC proliferation and pluripo-tency.[49] Synthetic materials have significantly advanced stemcell research and tissue engineering; however, implant rejectionremains challenging. New strategies were developed to promoteimmune tolerance without compromising functionality.[50]

Three-dimensional materials have been investigated for varioustypes of stem cell research. It would be interesting to developmaterials that are able to change stiffness over a period of time.We believe stem cell researchers would be thrilled to havematerials that provide a sequential release of growth factors overa period of time.

3. Engineering Concepts in Reprogramming

Tissue specific stem cells reside in organs which come to therescue when repair of the organ is needed. However, someorgans, such as the heart and pancreas, lack the presence ofthese resident stem cells for repair. Recent research directionsprovide hope for these repairs by applying knowledge gainedfrom developmental biology in the differentiation of pluripo-tent stem cells.

3.1. Reprogramming to Generate Induced Pluripotent StemCells

Prior to 2006, pluripotent stem cells referred to cells derivedfrom embryos, which posed problems due to ethical issues.Pluripotency transcriptional factors such as Oct4, Sox2, Lin28,and Nanog were identified; delivery of these transcription factors





to somatic cells by Takahashi and Yamanaka yielded inducedpluripotent stem cells (iPSCs).[51] Reprogramming has evolvedsignificantly over a short period of time (since 2006) andmethodology used to generate iPSCs has undergone evolution.iPSCs were successfully generated using recombinant proteins,micro RNA, non-viral methods, small molecules, and syntheticRNAs. Table 2 shows different methods and their efficiency ofgenerating iPSCs; many of these methods have been commer-cialized to encourage wide use. Patient specific reprogramminghas been developed using the methods described in Table 2 tounderstand diseases during the developmental stages.[52–54] Inspite of the availability of different methods to generate iPSCs, todate, none of these methods have been approved by the Food andDrug Administration (FDA). Footprint-free reprogramming isessential to obtain clearance from the FDA and other regulatoryagencies to clinically translate autologous reprogramming forregenerative medicine applications. In this decade, there hasbeen tremendous research on reprogramming of varioussomatic cells to either pluripotent stem cells or tissue specificcells. Improvements in methods and validation will bring hopefor many diseases to be cured. Recently, attempts were made togenerate iPSCs under Good Manufacturing Practice (GMP)using episomal vectors with human umbilical cord blood.[55]

Though iPSC technology has several advantages, recent studieshave questioned these advantages with respect to epigeneticmemory and immunological reactions.[56,57]

3.2. Direct Reprogramming

To overcome some disadvantages (teratoma formation) ofpluripotent stem cell based differentiation, alternative methodswere developed to directly convert one cell type to another celltype by skipping the pluripotent stage (direct reprogramming).

Directly reprogramming one type of cell to another type haswidespread application in regenerative medicine. The keyengineering concepts involved in gene editing via directreprogramming activities include targeted gene transfer. Onesuch type of gene targeting was achieved using the CRISPR/Cas9 system[58,59]; there are a variety of examples available in theliterature. One example is the direct reprogramming of

Table 2. List of methods used to create iPSCs, their efficiency, andfootprint of vectors used in reprogramming.

Method Days Efficiency (%) Footprint

Lentiviral[63] 20 0.4 Yes

Adenoviral[64] 25 0.0002 Yes

Sendai viral[65] 25 1 No (after 10 passages)

PiggyBac[66] 25 0.02 Yes

Minicircle[67] 28 0.005 No

Episomal[68] 20 0.02 No (after 12 passages)

Protein[69,70] 60 0.001 No

RNA[71] 20 4.4 No

Micro RNA[72] 14 10 Yes

Small molecules[73] 40 0.2 No

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fibroblasts to neuronal cells with neurogenic genes by activatingendogenous genes via chromatin remodeling.[60] Reprogram-ming was also successfully demonstrated by manipulating themechanical properties available to cells. Human embryonickidney (HEK-293) and Madin-Darby Canine Kidney (MDCK)cells were successfully reprogrammed to stem cell-like cells byculturing them on soft substrates.[61] Micro-patterning ofadhesion molecules to grooved surfaces significantly improvedthe reprogramming of somatic cells to pluripotent cells withenhanced epigenetic modifications indicating the existence of abiophysical-epigenetic axis.[62]

4. Stem Cells and Microfluidics

Microfabrication and microfluidics have been used for variouscell culture applications including embryonic stem cells (ESCs)and induced pluripotent stem cells (iPSCs). One advantage ofminiaturization is the reduced sample volume needed as wellas other reagent materials which is crucial for a variety ofclinically relevant samples. Several materials have been usedfor microfluidics applications, such as polymers, plastics, andmetals. Of these materials, polydimethylsiloxane (PDMS) isone of the most popular owing to its physical, chemical, andoptical properties and its low cost. Some salient features ofmicrofluidic cell culture devices include continuous flow ofmedia, which provides a microenvironment with a constantsupply of growth factors and other chemical molecules that arerequired for cellular activity. Microfluidic devices for cellculture constantly eliminate the waste generated by the cellswhile, in conventional static cell culture systems, the wastebuilds up locally and affects cell physiology. Microfluidicdevices with perfusion systems often use conventional syringepumps; however, such systems are cumbersome because ofthe various tubes and connectors. To improve upon the syringepump, pressure-driven perfusion systems were developed. Thepressure exerted by the liquid reservoir helps to drive perfusionof the reagent into the microfluidic device. Such systems wereused in the determination of IC50 of paclitaxel in a perfusionsystem.[74]

The fluid in the microfluidic device can be manipulated in avariety of ways such as capillary driven, centrifugal driven,elctrokinetic, droplet based, acoustic wave based, and inertialmicrofluidics. Apart from these manipulations, the physicaldensity of a liquid plays a role in the separation of biologicalmaterials using centrifugal force. Microfluidic droplets havebeen put to use in various stem cell based applications to studystem cell heterogeneity.[75] In most cases, the cells were analyzedafter encapsulation, because of the limited oxygen and nutrientsin the droplets. To overcome oxygen depletion, oxygenpermeability was enabled through the device or via fluorinatedoils.[76–78]

4.1. Microfluidics and Stem Cell Culture

Gene expression analysis is a common method used in theidentification of stem cells and related lineages. Many geneexpression profile studies in stem cell research use





heterogeneous cell populations as a default and these results aredifficult to interpret sufficiently for relative percentages of stemcells and progenitor cells. Single-cell gene expression profilingwill provide reliable information on critical regulator factorsinvolved at a specific stage of cell proliferation or differentiation.Single-cell transcriptome analysis has been proposed to preciselyunderstand all biological information happening in a single cell.Microfluidics drastically reduced the conventional requirementof cell sorting or laser capture for gene expression analysis.Zhong et al., constructed a device to capture 20 single hESCs andanalyzed expression profiles of beta-2-microglobulin, Nodal andFrizzled-4; results indicate broad differences between conven-tional heterogeneous analysis and single-cell analysis.[79] Tran-scriptome analysis at the single-cell level will give preciseinformation on transcriptional activities (both coding andnon-coding) at a specified stage cell physiology (such as cellcycle). Klein et al. have used a droplet based microfluidic deviceto encapsulate individual mouse embryonic stem cells (mESCs);results obtained from this study helped to deconstruct theheterogeneity during stem cell differentiation.[75] Apart fromgene expression analysis, genetic manipulation at the single-celllevel was attempted usingmicrofluidics. Valero et al. developed amicrofluidic based electroporation device to genetically modifyhMSCs and demonstrated successful transfection of the greenfluorescent protein gene into each hMSC.[80] This type of deviceis advantageous to generate a homogenous cell population withthe same copy number of the gene of interest which isimpossible to obtain in conventional transfection/transductionmethodologies. The bimolecular gradient is an essentialcomponent in developmental biology. Stem cells createbimolecular gradients by secretion of growth factors andsignaling molecules. These gradients play a critical role indefining the phenotype of differentiating cells. Generatinggradients in microfluidics offer a wide range of control andprediction of a precise concentration dependent effect on stemcells.[81] Chung et al. have developed a gradient system to growneural stem cells and studied their proliferation and differentia-tion properties in the presence of a growth factor cocktail(epidermal growth factor, fibroblast growth factor 2, and platelet-derived growth factor). Apart from growth factors, gradients canbe made with chemicals (small molecules) and oxygen to studytheir effect on stem cell proliferation and differentiation.[82]

4.2. Microfluidic System for Circulating Tumor Cells (CTCs)and Cancer Stem Cells (CSCs)

Tumor cells may circulate in blood due to their metastaticcharacteristics. These circulating tumor cells (CTCs) are thehallmark of the invasive behavior of cancer cells and are used as aprognostic marker for cancer. The isolation of CTCs has severaladvantages; it enables us to perform functional assays (xeno-graft) and helps us to understand the biomolecular characteri-zation of these cells, treatment modality estimation, and diseaseprogression. CTCs are rare cells; detection and isolation of CTCsrequire highly sensitivemethodologies combined with the abilityto detect single cells. The majority of microfluidic devices utilizethe size difference between CTCs and blood cells to detect andisolate CTCs; however, othermethods such as dielectric potential

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(DEP) and antibody based capture were also utilized to identifyCTCs.

Droplet microfluidics is one technology used in single-cellanalysis. Droplet microfluidics hold great potential in drugdiscovery and single-cell omics. The droplet methodologyenabled analysis of rare cells from a large cell population andhence has promising bio-pharmaceutical application with highthroughput screening and validation of drugs. One suchapplication is the isolation of rare cells, especially cancer stemcells from various biopsy samples and cancer patient blood.Circulating cancer stem cells (CSCs) provide crucial informationfor clinicians as well as basic science researchers to understandthe stages of cancer and the phenotypic nature of cancer. It is alsostrongly believed that the CSCs provide essential information onresistance to existing therapies and possibilities of recurrence ofcancer. Several methodologies have been developed to isolateCSCs that involve multiple steps in the isolation procedure.[83–85]

Low purity complicates downstream applications; hence, ahighly sensitive and simple procedure was developed usingmicrofluidics.

Sarioglu et al. used microfluidic chips to capture circulatingtumor cell (CTC) clusters directly from the patient’s bloodsample without additional processing. The device consists ofthree triangular pillars; the CTC clusters are held at the edge ofthe bifurcating pillar. The flow speed and other parameters arecontrolled to maintain the integrity of the cell clusters. The keyfeature of this device is the direct processing of blood sampleswith no additional labeling required to isolate the clusters.Cluster trapping and isolation is mainly achieved through thegeometric nature of the cells and the triangular pillars.[86]

Microfluidics is also used in macromolecular delivery into thecells; intracellular delivery of cargo is better understood,specifically with membrane disruption methodologies. Tran-sient disruption of the membrane is the key for successfuldelivery of cargo and this knowledge has influenced designconcepts to develop microfluidic channels to squeeze cells withdelivery cargo. Interestingly, Sharei et al. used such a device toreprogram somatic cells.[87] Recently, Kim et al. have combinedimmunomagnetic nanobead based CTC isolation with droplettechnology to yield droplets containing single CTCs.[88] Fewmicrofludic devices for CTC isolation have been successfullycommercialized as shown in Table 3. Microfluidic devices havehelped tremendously in advancing CTC research; however,similar devices in pluripotent stem cell research are still in theearly stages of development. Improved and validated micro-fluidic systems will bring significant advancement to pluripotentstem cell research.

5. Nanotechnology for Stem Cells

Nanotechnology tremendously aided in understanding biologi-cal interactions at the nanoscale level, be it cell-biomaterial orcell–cell interactions. Applying nanotechnology to biologicalsystems, especially in genemanipulation of stem cells, has directimpact on influencing the therapeutic benefits of stem cells. Inshort, nanotechnology allows us to identify stem cells,manipulate individual cells and modify their niche to generateclinically relevant cells.




Table 3. Table lists microfluidic devices successfully commercialized and methods used in these devices.

Size DEP Surface marker (SM) SMþDEP SMþ size

Method Size and deforming property, arrays

of pillars or micro sieve[89–91]Differential flow

and retention[92,93]Anti-EpCAM conjugated to either magnetic

beads, nanopillars or microposts[94–96]Electromigration or

magnetic

beads in combination

with anti-EpCAM[97,98]

Microposts coated

with anti-EpCAM[99]

Companies Clearbridge biomedics ApoCell CytoScale diagnostics BioFluidica On-Q-ity Inc


5.1. Nanotechnology and Stem Cell Microenvironment

Tissue engineering is an emerging interdisciplinary field,combining the principles of biology, engineering, and materialscience as well as medicine, to create an implantable biologicalsubstitute to restore, replace, and maintain/enhance thefunction of damaged tissues and organs. Within the tissueand organ, cells are surrounded by the nanostructuredextracellular matrix (ECM) and directly interact with the nano-sized macromolecular organic components of ECM. Thisinteraction via cell-ECM is important in terms of maintainingthe functionality of the cells as ECM provides a comfortableniche for the cell. Over the past few years, remarkable success inthis field has resulted in the establishment of implantabletissues; some are already implanted into humans, such as skinand cartilage, and a few are in the clinical trial phase, such asbladder and blood vessels.[100] Biomimetic 3D scaffolds havebeen used in a tissue engineering approach to develop newtissue for transplantation.[100,101] A recent advancement in thefield of nanotechnology has formulated biomimetic scaffolds atthe nanoscale size, which are capable of mimicking the tissue-specific microenvironment to regulate and maintain cellfunction and behavior.[102] There are various methods tofabricate scaffolds with an extracellular matrix-like architecturesuch as electrospinning, nanopatterning, self-assembly, phaseseparation, and conjugation of adhesion motif or sulfating thematrix backbone.[103,104] Fabrication of nanofibers by electro-spinning offers attractive properties such as affordability, highsurface/volume ratio, availability of a wide range of materials,low amounts of initial solutions, and diameters in the range of3 nm to several micrometers. Owing to these properties,electrospinning is the most extensively used method.[105] Todate, scientists have made scaffolds by using different nano-materials such as carbon nanotubes, nanofiber, nanowires, andnanoparticles (titanium, gold, etc.).[106,107] In developing nano-materials, biomimic ECM’s structural, and nano-topography hassignificantly advanced our understanding of various cellularactivities of stem cells during expansion and differentiation.Cell–cell interactions and cell–material interactions with differ-ent nanomaterials have been studied with nanopatterningtechniques.[108,109] In the case of hMSC adhesion and differenti-ation to an osteogenic lineage, Park et al. demonstrated thatvertically aligned TiO2 nanotubes with spacing of 15 nm enabledsignificant cell attachment and differentiation compared to100 nm spaced nanotubes.[110] The authors have carefullycalculated spacing of integrins on the cell surface and indicateda close resemblance to 15 nm spacing.

A variety of nanotechnology methods based on differentcomponents of tissue engineering such as biomaterial scaffolds

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and biomolecules (ECM, growth factors, and other functionalmolecules) have been implemented. Nanotechnology in tissueengineering offers benefits over conventional methods and ameans todirect cells fromanadhesionmolecule to themodulationof gene expression. The interaction between cells and theirsurroundingmicroenvironment at the nanoscale is specific to celltype and it regulates the cellular signaling network whichdetermines cellular fate. The biomimicked scaffolds can be tunedwith mechanical, biochemical, and electrical properties that aresimilar to native tissues.[111] Stem cell differentiation into aparticular lineage is possible using scaffolds containing specificbioactive molecules or topographies.[112] Jia et al. fabricated anelectrospun nanofiber with poly-L-lactide (PLLA) and hybridPLLA/collagen (PLLA/Coll) (3:1 and 1:1) with fiber diameters of210 to 430nm for the differentiation of bone marrow derivedmesenchymal stem cells (BM-MSCs) into endothelial cells (ECs).They concluded that this technique was promising for vasculartissue engineering.[113] Additionally, scaffolds for fabrication and/or regeneration of body organs (such as heart, liver, and kidney)and the nervous system (including the spinal cord and brain) havebeen developed.[114] Akhavan et al. developed rolled grapheneoxide foam (GOF) layers as an electrical conductive 3D scaffold fordifferentiation of humanneural stemcells (hNSCs) that are usefulin neural tissue engineering. Due to the low electric sheetresistance of GOFs under low voltage (<5V), the electricalstimulation current produced was suitable for the differentiationof hNSCs into neurons. They reported enhanced proliferation ofthe cellsandaugmenteddifferentiation intoneurons, ascomparedto glia.[114] Similarly, Bhaarathy et al. created a nanofiber scaffoldfor cardiac tissue.Toobtain scaffoldwithdesirablemechanical andfunctional properties, they developed a scaffold by electrospinningcopolymer Poly (L-lactic acid)-co-poly (e-caprolactone) (PLACL),silk fibroin (SF), and Aloe Vera (AV). PLACL/SF/AV nanofibersshowed good regeneration of infarcted myocardium and expres-sion of cardiac proteins such as myosin and connexin.[115]

Interestingly, for cartilage tissue engineering, Lim et al.developed a dual growth factor, bone morphogenetic protein-7(BMP-7) and transforming growth factor β2 (TGF-β2), loadednanoparticle/hydrogel system for the controlled release of BMP-7/TGF-β2. This system provides the required growth factordelivery kinetics for cartilage regeneration, as well as thechondrogenesis of MSCs, i.e., fast release of BMP-7 but slowrelease of TGF-β2.

[116] However, in another study by Lam et al. onhuman embryonic stem cell (ESC)–derived neural cells, abiodegradable poly(l-lactic acid) scaffold adsorbed with growthfactors such as basic fibroblast growth factor (bFGF) andepidermal growth factor (EGF) have been used for neural tissueengineering. The growth factors attached onto nanofibersthrough heparin were advantageous as compared with absorbed





growth factor. The heparin conjugated scaffold with bFGF andEGF helps to preserve their bioactivity and increase their half-lifeand also significantly promotes neural differentiation and axongrowth for neural tissue regeneration.[117,118] In addition tophysical cues andmacromolecular cues, chemical cues were alsostudied for their role in cellular activity of stem cells. Forexample, Li et al. demonstrated that positively (amine) chargedgold nanoparticles (AuNPs) increased proliferation of hMSCswith increased expression of FGF-2 in comparison withnegatively (COOH) charged AuNPs.[119] This finding introducesa new dimension to stem cell behavior in functionalizednanomaterial and warrants a comprehensive study.

5.2. Nanotechnology and Stem Cell Manipulation

Uptake of nanoparticles (NPs) under physiological conditionshas enabled the use of NPs for genetic manipulation of cells. Avariety of NPs has been used extensively for delivery of DNA,mRNA, small interfering RNA (siRNA), microRNA (miRNA),and proteins. NPs were used to overcome the adverse effects ofviral vectors. Polymeric NPs (PLGA based) have been used todifferentiate hMSCs to chondrocytes. SOX9 coated PLGA NPswere shown to induce differentiation of hMSCs with increasedexpression of chondrogenic genes (type 2 collagen, aggrecan,cartilage oligomeric matrix protein).[120] Similar materials havealso been used to differentiate hMSCs to adipocytes with PLGANPs coated with two genes (CCAAT/enhancer binding pro-teins).[121] Other polymers such as chitosan and palmitic acidwere used for gene transfection in hMSCs.[122] Superparamag-netic iron oxide NPs were used to deliver the brain-derivedneurotrophic factor (BDNF) gene in hMSCs. Transfectionefficiency was higher compared to polymer (PEI) based deliveryand transfected cells were functional in a rat sciatic nerve defectmodel.[123] Green et al. developed strategies to deliver the greenfluorescent protein (GFP) gene to hESC clusters. The authorsused 200 nm, positively charged polymeric biodegradable NPs todeliver the GFP gene and noticed that efficiency was four timeshigher than conventional Lipofectamine 2000 transfection.Additionally, NP mediated gene delivery had minimal cytotoxic-ity and reduced non-specific differentiation.[124] Apart from geneactivation, gene silencing was performed with NPs coated withsiRNA. Huang et al. developed a system for near infrared (NIR)light mediated gene silencing in hESCs. The authors havesuccessfully inhibited a pluripotent gene, Oct4 with Oct4 specificsiRNA leading to differentiation of hESCs into three germlayers.[125] Such a system with inducible gene silencing would beof interest to stem cell researchers. In spite of great progress ingene delivery with NPs, there are still several issues that limit itsuse. One such important question that needs to be addressed isthe degradability of NPs; the fate of NPs and the by-productscreated clearly limit its use in humans.

5.3. Nanotechnology and Stem Cell Tracking

The success of cell based therapy relies on the functionalcapability of implanted cells. Tracking and monitoringimplanted cells is key for the ultimate success of cell based

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therapy.Withminimal invasiveness, NPs have been used to trackstem cells via labelling and visualization of the implanted cells atthe site of action. Methods commonly used in labelling andtracking of stem cells include magnetic NPs, quantum dots(QDs), AuNPs, carbon nanotubes, and nanodiamonds.[126] NPsrequire functionalization and coating for cellular uptake. Forexample, superparamagnetic iron oxide (SPIONs) were coatedwith dextran for labeling hMSCs; dextran coating helps with theinternalization of NPs. SPION labeled cells can be imaged usingmagnetic resonance imaging (MRI).[127] In addition, SPIONscan be coupled with specific antibodies, proteins, and drugs tomanipulate implanted cells.

Cell therapy with MSCs augments regeneration of skeletalmuscle in a muscle injury model; additionally, retention of stemcells at the site of injury potentially improves regeneration.Magnetic cell labelling methods using an external magnet, non-invasively, allows for guidance, and retention of stem cells at thesite of action. Site-specific targeting was demonstrated withhMSCs in nude rats.[128] SPIONs have been successfully used intracking MSCs in different species such as rat,[129] porcine,[130]

canine,[131] and ovine.[132] Another method of stem cell labellinguses QDs with tunable excitation and emission properties. QDsare routinely functionalized with peptides to help in cellpenetration and endosomal escape. Some of these peptidesact as cell penetrating peptides and others help in the endosomalescape of QDs. Slotkin et al., used Qdot-620 to label neural stemand progenitor cells (NSPCs) and microinjected them intodeveloping mouse embryos in utero. The authors were able totrack injected cells in the form of differentiated cells such asastrocytes, oligodendrocytes, and neurons.[133]

Understanding biological systems at the nanoscale leveldelivers unique biophysical and biochemical composition, whilemicro and macro engineering methodologies help us to developfunctional tissue constructs (Figure 2). The nanotechnologicalapproach offerswide availability ofmaterialswith tunable featuresand biomolecular macromolecule loading for successful use instem cell research and regenerative medicine. We need verticalresearch on some of thesematerials to understand their long-termeffects andmetabolic influences in stemcells and theirderivatives.Nanotoxicity is an extensive area of research which warrantssignificant focus to develop clinically relevant nanomaterials.

6. Bio-Printing Stem Cells

Recent advancements in additive manufacturing technologiesand emerging needs in biomaterial development have helped toevolve the concept of 3D bio-printing using bioinks. Stem cellsare popular for developing 3D printed living tissue as theyenhance tissue engineering for in vitro modeling andregenerative medicine.[134] Bio-printing manages the develop-ment of functional and viable cell patterns arranged in aconfined space using 3D printing technologies.

6.1. Design Approaches

Stem cell based bioink, when printed as tissue, is supposed todifferentiate in situ into functional cells together with




Figure 2. Schematic representation of nanotechnology application in stem cell research.


supporting cells or ECM.[135] Figure 3 describes the stepsinvolved in development of a 3D printed functional tissue/organ.[136] Unlike 2D monolayer cell culture, robust designapproaches are needed which can better mimic the natural tissueenvironment and homogeneous/heterogeneous interactionsamong the cells and building blocks. To conceive biomaterialswith these parameters, various design approaches includingbiomimicry, autonomous self-assembly and mini-tissue devel-opment are available, which act as a foundation layout for 3Dprinting.[136] The biomimicry approach focuses on the develop-ment of the tissue or organ mimicking the shape, framework,and microenvironment of the natural structure. The stem cellsare well versed in autonomously organizing into the requiredbiological architecture and function together with the ECM;thus, they are compatible with the autonomous self-assemblyapproach. A common hybrid approach in mini-tissue develop-ment which involves both previously mentioned approaches. Itprovides functional building blocks of elemental structureswhich can be assembled together as bio-printed tissue.[137]

Figure 3. Three-dimensional (3D) bio-printing of stem cells usingbioinks. Steps involved in preparation and development of bio-printedorgans from stem cells.

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6.2. Stem Cells With Bioink

The sourcematerial for 3D bio-printing is comprised of syntheticor natural polymers and ECM together with live cells, which canbe functionally differentiated cells or stem cells. A well-definedpatterned structure of tissue is obtained when this bioink isdeposited layer-by-layer with a 3D printer.[138] The gel type bioinkcross-links and provides a porous 3D platform for in situ growthand differentiation of stem cells. Lee et al.[139] printed artificialneural tissue of C17.2 murine neural stem cells by developingcell-collagen hydrogel composites. The vascular endothelialgrowth factor (VEGF) containing fibrin gel was printed using a3D bio-printer together with the cells, where the gel helped withsustained release of growth factors in the collagen scaffold. Rutzet al.[140] demonstrated a multi-material approach for 3Dprinting tunable and cell-compatible hydrogels as bioinks. Theyused various amine-containing polymers and their mixtures andoptimized different parameters of printability together withstructural and biological performance. Stem cell bio-printingprovides a promising solution for peripheral nerve defect repairas demonstrated by Hu et al.[141] who worked with acryopolymerized gelatin methacryloyl (cryoGelMA) gel basedbio-conduit cellularized with adipose-derived stem cells (ASCs).The cryoGelMA scaffold supported the seeded ASCs andupregulated neurotrophic factors of mRNA. The model workedwell as cellularized conduits for peripheral nerve regeneration ina rat model.

Gu et al.[135] developed polysaccharide-based bioink consist-ing of alginate, carboxymethyl-chitosan, and agarose and used3D extrusion direct-write bio-printing together with humanneural stem cells. The bioink with the capability of cross-linkingstably and converting into porous 3D scaffolds supported theproliferation and differentiation of neural stem cells in situ intofunctional neurons and supporting neuroglia. They furtherdemonstrated the functionally active synaptic contacts in the





developed bio-printed tissue, which is valuable in studying andcharacterizing neural development and functions.

Armstrong et al.[138] reported a novel pluronic-alginatemulticomponent bioink in a two-step 3D printing process togenerate bone and cartilage tissue. They printed humanmesenchymal stem cells (hMSCs) together with the gel, whichprovided promising biomaterial properties such as increasedshear thinning, compressive modulus, and shear modulus. Inaddition, they have shown that the encapsulated stem cells canbe differentiated into osteoblasts and chondrocytes, furtherdeveloping the tissue as a tracheal cartilage ring. Similarly,Nguyen et al.[142] printed human iPSCs into cartilage using ananofibrillated cellulose (NFC) composite bioink together withirradiated human chondrocytes. These bio-printed tissues arepaving the path for novel treatments to repair damaged cartilage.

6.3. Bio-Printing Strategies and Challenges

For 3D bio-printing, surface resolution, cell viability, and bioinkused for tissue generation are among the primary factorsconsidered when defining the strategy for deposition and patterngeneration. Based on these factors, bio-printer technologies likeinkjet bio-printing, micro-extrusion, and laser-assisted printingare available.[136,143]

Bio-printing is a complex interdisciplinary field whichdemands advanced technologies in the fields of engineering,biomaterials science, chemistry, cell biology, physics, andmedicine. The delicate viability of stem cells demands thatthe choice of biocompatible materials, cell proliferation, anddifferentiation environment must be considered together withsupporting cells and ECM during the course of 3D printing andincubation into a developed tissue. Also, a major challenge is toincorporate functional characteristics and desirable cell-to-cellinteractions. Although it is challenging, the technologicaladvancements are driving the research community to closethe gap and overcome these hurdles.

7. Future Directions

Stem cells with plasticity are a key tool in regenerative medicine.Research with stem cells can be grouped into the followingdivisions; 1) to develop differentiation strategies; 2) to study thepathophysiology of diseases; 3) to discover new drugs; 4) todevelop cell based therapies; and 5) to reprogram somatic cells.Both adult and pluripotent stem cells contribute at differentlevels for the advancement of regenerative medicine. Despitebeneficial outcomes with several technologies to advance stemcell research discussed in this review, further advancement forsuccessful clinical translation of these findings is still needed.For example, stem cell differentiation protocols with highefficiency yields of specific cell types still harbors a small fractionof undifferentiated stem cells or other cell types, leading toteratoma formation. Isolation of differentiated cells for celltherapy applications is a requirement and it is achievable withsome of the technologies described herein. For cell therapyapplications, a large number of cells are required and futureresearch potentially should focus on the industrial culture of

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stem cells and their progenies. Additionally, monitoring “stem-ness” during such large scale culture system requires constantreal-time monitoring methodologies with simplified biomarkersand assay systems. Reprogramming somatic cells has created aspecific niche and numerous studies have been reported.Current stem cell research trends focus on reprogrammingsomatic cells; direct reprogramming to cell-type of interest skipsthe pluripotency path, however, with limited efficiency andefficacy. Stem cell research at the micro or nano scale gives us aclear understanding at the cellular level; however, translation ofthese findings requires macro constructs leading to a completefunctional tissue. The success of the tissue engineeredmulticellular organ relies on the key factor of vascularization.Vascularization is the downfall of many products that fail duringdevelopment. Awide variety of technological advances have beenmade in past decades to incorporate various engineering aspectsinto biological systems; however, major challenges exist withtransferring these technological developments into clinicalapplications. For example, there has recently been substantialwork performed on spheroids or organoids; however, clinicalapplication of these organoids has not yet been demonstrated,primarily because of the lack of methods to connect theseorganoids to form a complete organ. In the past few decades,engineering concepts have helped to develop and design betterbiomaterials, microfluidic chips, and bio-printing. Engineeringideas in biology will benefit mankind by reducing the cost ofmanufacturing and the alliance between science and engineer-ing will continue to overcome the challenges that exist in thetissue engineering field.

AcknowledgementsSachin Mishra and Satnam Singh contributed equally to this work. Thiswork was supported by Research Grants from the MusculoskeletalTransplant Foundation and the National Institutes of Health(R03AR062763-01A1, R01AR067747-01A1) (to M.P.). The Authors (PPand BG) acknowledge the support from Lee Kong Chian School ofMedicine, Nanyang Technological University Start-Up Grant. We wouldlike to thank Ms. Suzanne Danley (Department of Orthopaedics, WestVirginia University) for editing the manuscript.

Conflict of InterestThe authors declare no commercial or financial conflict of interest.

Keywordsbioengineering, biomaterials, bio-printing, microfluidics, regenerativemedicine, reprogramming, stem cells, tissue engineering

Received: May 28, 2017Revised: September 7, 2017

Published online: September 25, 2017

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