Human embryonic stem cell cultivation: historical perspective and evolution of xeno-free culture systems

One of the first approaches tested for feeder-free growth relied on the use of extracellular matrix proteins in combination with growth factor supplementation to create an in vitro microenvironment that promoted stem cell renewal. The extracellular matrix, Matrigel isolated from mouse Engelbreth-Holm-Swarm teratocarcinoma cells was used by Xu et al. [6] as a feeder cell substitute for hESC culture. Matrigel is a gelatinous mixture of collagen IV, laminin, proteoglycans, entactin and other growth factors [56]. To date Matrigel, often in combination with growth factors or conditioned medium from feeder cultures has been the most frequently used system for hESC culture [57-63]. Lot-to-lot variations in the Matrigel composition can however pose problems, inhibiting its effectiveness in maintaining cells in a pluripotent state, thus affecting reproducibility of subsequent stem cell differentiation protocols. The use of Matrigel also raises clinical concerns as certain batches have been found to be contaminated with the single stranded mouse RNA virus, Lactate Dehydrogenase Elevating Virus (LDEV), ultimately questioning the safety of using stem cells that have been cultured on animal derived extracellular matrix in a therapeutic setting [64].

Fu et al. extracted the extracellular matrix from embryoid bodies (e-ECM) derived from the H9 ESC line [45]. This human-derived ECM consisting of fibronectin, laminin and collagen type IV, proved to be a viable method for xeno-free propagation of hESCs used to generate a xeno-free autologous feeder-free culture system for long term culture of the H9 hESC line. The hESCs were successfully maintained for 20 passages on this ECM substrate in TeSR2, a chemically defined serum-free and animal protein free medium [65,66]. Spontaneous differentiation in hESCs cultured on e-ECM was markedly less than on Matrigel, suggesting a more supportive environment. This xeno-free culture model was also successfully used for terminal differentiation of the hESCs in to keratinocytes for potential therapeutic application [67]. Human placenta–derived ECM has also been successfully used for hESC culture [68]. Cultures showed genetic stability after 40 passages and differentiation potential of the stem cells was retained.

These studies with extracellular matrix have been instrumental in identifying biomolecules involved in stem cell renewal. Inconsistencies in ECM preparations even if of human origin, do however still present a problem. Certainly, use of chemically defined matrices and recombinant proteins based on ECM may be a more reliable approach towards xeno-free culture of therapeutic grade hESC lines. Xu and colleagues analyzed the individual components of Matrigel and determined that not all are equally effective for sustaining undifferentiated growth of ESC lines [6]. Laminin appeared to be superior to fibronectin and collagen IV for feeder-free growth of stem cells in MEF-conditioned medium [6]. Others have successfully used human fibronectin with bFGF and TGFβ to culture hESC for over 180 days [69]. Beattie et al. [51] showed that medium supplementation with Activin could be used to eliminate the need for MEF cell conditioned medium. Successful propagation of hESCs in a serum and xeno-free medium, TeSR1 using a combination of human matrix components collagen IV, laminin, vitronectin and fibronectin as a substratum marked a significant milestone in the development of clinically compliant hESC culture models [66,70]. This culture system allowed derivation of a karyotypically normal hESC line (WA-15) and also the undifferentiated propagation of H1 and H9 stem cells under defined conditions without animal products. Inclusion of Rho kinase inhibitor (ROCK) in the serum free medium helped prevent apoptosis in individual cells at passaging.

Laminin with its role in cell adhesion has been extensively studied. Rodin et al. described a xeno-free system for hESC and hiPSC cultivation using a recombinant form of human laminin-511 with a defined medium, TeSR1 [71]. This serum-free culture system allowed hESC cell renewal with genetic stability for 4 months (20 passages). Miyazaki tested different recombinant human laminin isoforms for their ability to serve as scaffolds for hESC cultivation [72]. Laminin isoform binding to integrin receptors α6β1 on hESC appeared to be integral to triggering signaling pathways necessary for continuous self-renewal. Recombinant human laminin isoforms LM- 111, 511 and 332 supported hESC adhesion and undifferentiated cell growth. Further studies showed that even fragments of different recombinant laminin isoforms (LM E8’s) if they contained integrin binding capacity could be used for hESC culture [73]. LM 511-E8 was in fact found to be superior to Matrigel or whole laminin isoforms in promoting cell adhesion and proliferation of stem cells. Stem cells could be cultivated under xeno-free conditions in a chemically defined medium (TeSR2) on the LM511-E8 coated substrate for over 30 passages and retained pluripotency as well as a karyotypic stability. The smaller size of LM-8 s as compared to whole laminin simplifies manufacturing the recombinant product making it more cost-effective. Nakagawa and colleagues reported better attachment of hESC and hiPSC dissociated in to single cells using StemFit media with laminin 511-E8 matrix [74].

E-cadherin, another cell adhesion molecule that binds to the integrin α6β1 also plays a role in cell:cell binding [75]. Poor survival and hESC death after dissociation into individual cells is due to a disruption of E-cadherin signaling [76-78]. Nagaoka et al. cultured hESC and hiPSC cells on plates coated with E-cadherin fused with the IgG FC domain [79]. This feeder-free system with a defined medium, TeSR1 was effective as a substitute for Matrigel. The matrix promoted adhesion and both types of pluripotent stem cells (hiPSC and hESC) retained their stem cell attributes for over 90 days in culture. Supplementation of culture media with E-cadherin in combination with recombinant laminin isoform 521 increased efficiency of hESC line derivation from individual blastomeres as well as the ICMs of blastocysts in chemically defined media under xeno-free conditions [80]. This vital work provides a clinically compliant system for the derivation of new stem lines for regenerative medicine. The E-cadherin/laminin 521 based culture system also allowed robust proliferation of hiPSC cells under xeno-free conditions. A cocktail of specific kinase inhibitors along with bFGF may also be beneficial for propagating single stems cells under feeder-free chemically defined conditions [81].

Amongst ECM components, vitronectin is another protein that has emerged as a promising substratum for long term culture of hESC cell serum-free conditions in defined media [82-85]. Recombinant vitronectin was able to effectively replace Matrigel as a substrate for hESC culture in a defined medium [82]. Chen et al. devised a simplified culture system for hPSC using a truncated recombinant vitronectin coating and a chemically defined, xeno-free and albumin-free medium known as Essential 8 (E-8) [83]. This represents a significant advancement in the movement towards clinically compliant culture models for stem cell applications. Hasegawa et al. [84] identified two small chemical molecules that could replace bFGF in culture media and still sustain human stem cell growth [84]. These molecules are modulators of the Wnt/β-catenin signaling pathway [86-88]. Based on molecular clues present in the environment, this pathway can drive hESC cells towards continued cell renewal as well as differentiation. The combination of Wnt modulators with ECM-based substrata (vitronectin, laminin or fibronectin) allows culture in a chemically defined media without exogenous supplementation with bFGF or TGFβ.

Synthetic substrata for stem cell culture have also been investigated as alternatives to complex ECM matrices. Synthetic peptide acrylate surfaces (PAS) constructed by conjugating peptides derived from the biologically active region of extracellular matrix proteins can sustain the self-renewal and pluripotency of hESCs for over 10 passages in a xeno-free defined medium X-Vivo 10 [89]. In this study, Melkoumian et al. tested acrylate surfaces with bound peptides from laminin, vitronectin, fibronectin or bone sialoprotein. PAS with vitronectin or bone-sialoprotein derived peptides showed the best functionality. These cultured stem cells were also capable of directed differentiation in to cardiomyocytes. Corning Synthemax Surface, a synthetic acrylate surface with RGD peptide containing sequence from vitronectin was shown to support the expansion and directed differentiation of human induced pluripotent cells in defined medium [90]. More recently, investigators demonstrated that Synthemax could also be used for the efficient expansion of not only colonies but also single dissociated hPSCs in several serum-free and xeno-free culture media like TESR2, Nutristem XF and PSGro [91].

Klim et al. [92] systematically screened 18 bioactive peptides bound to different substrata to identify peptide surfaces that could sustain hESC growth under defined conditions. Surfaces with the heparin binding pepetide GKKQRFRHRNRKG isolated from vitronectin were shown to facilitate hESC adhesion and proliferation [92]. This peptide was demonstrated to function by interacting with hESC cell- surface glycoasoaminoglycans. Interestingly, surfaces prepared with only peptides containing the core integrin binding motif (arginine-glycine-aspartic acid, RGD) were inadequate for sustained hESC culture.

Wu et al. recently described a novel synthetic material based on spider silk proteins as a suitable substrate for hESC culture [93]. This artificial matrix composed of recombinant miniature spider silk protein, 4RepCT (~3000 base pairs) can be easily manufactured and formed into films, fiber or foam under sterile conditions. Variants of the matrix were created by fusing it with biological peptides derived from laminin or vitronctin. The investigators cultured both hESC and hiPSC lines on 4RepCT-vitronectin films in xeno-free defined medium Nutristem for over 30 passages without loss of stem cell attributes. The cultured hPSC had the capacity to form teratomas in vivo and to undergo directed differentiation into endoderm and also cardiomyocytes in vitro. The properties of the biomatrix allowed for 2-D as well as 3-D culture.

The cost of peptide synthesis, stability of substratum with attached biological molecules and anticipated difficulties with scale up during manufacture has triggered interest in establishing purely synthetic surfaces for serum-free culture of hESC. This has proved to be particularly challenging. Numerous polymer based synthetic surfaces such as PMEDSAH (poly [2- (methyacryloyoxy) ethyldimethyl-(3-sulfopropyl) ammonium hydroxide]), PMVE-alt-MA (poly (methyl vinyl ether-alt-maleic anhydride)), HIT9 and APMAAm (aminopropylmethacrylamide have been tested [89,94-99]. Amongst the tested synthetic polymers, PMEDSAH [98] and APMAAm [95] appear to have the most promise, supporting growth of several hESC lines for at least 20 passages with retention of pluripotency and karyotypic stability.

Transition towards xeno-free culture systems has necessitated the formulation of new media suitable for cultivation of stem cells under serum-free conditions [66,70,83,100,101]. Basal medium such as DMEM/F12 containing amino acids, glucose, vitamins, insulin, transferrin, selenium in a balanced salt solution has been the foundation for many of the in-house as well as commercially available culture media for hESC cultivation. Cholesterol, lipids, GABA, pipecolic, ascorbic acid and β mercaptoethanol are also often included. The compositions of several commercially available media are shown in Table 3. In the absence of serum, all either contain or require bFGF supplementation at concentrations ranging from 20–100 ng/ml. Other select growth factor/cytokine additives used in the various media formulations are TGFß, Activin A, LIF, stem cell factor (SCF) and a structurally similar cytokine FLT3 (FMS-like tyrosine kinase). Under xeno-free conditions, suppression of signaling by bone morphogenetic protein (BMP) can prevent loss of stem cell pluripotency [59,62]. BMP-antagonists like Noggin when included in culture media work synergistically with bFGF to maintain stem cell homeostasis. Another inhibitor of BMP signaling, dorsopmorphin along with inhibitors of the Wnt signaling pathway also work to suppress spontaneous differentiation in pluripotent cells in a xeno-free culture medium [102].

Commercial protein supplements like KnockOut –Serum Replacement (KSR) are often used in culture systems to replace fetal bovine serum but do in fact still contain animal derived serum albumen. Only a few of the media described are completely xeno-free with human serum albumen (HSA) as a protein source in lieu of bovine serum albumen (BSA). However, since HSA is extracted from different pools of donor serum there are batch-to-batch variations. Also HSA manufacturing guidelines only require a purity of ≥96% so most commercial preparations for cell culture contain other undefined plasma proteins and contaminants from the purification process. To technically be classified as a “chemically defined medium”, all constituents of the medium and their concentrations must be known [103,104]. Media formulations with BSA or HSA extracted from plasma, introduce contaminants that may not always be precisely controlled and therefore do not meet this strict definition. Confusion over this terminology is evident from the literature. Numerous media that were developed with bovine and/or human albumen in lieu of serum have early on been referred to as “defined or “chemically defined” (as compared to serum containing media), if all other media components were quantifiable. Recombinant HSA is now often substituted for serum derived HSA in designing new chemically defined media. Table 3 describes commercially available media for serum-free culture of hESCs and information on their composition. Several xeno-free chemically defined media are currently on the market such as TeSR2 and PSGro with recombinant HSA, as well as Essential 8 (E8) and TESR-E8 which do not contain albumen. X-Vivo 10, a defined medium, with recombinant growth factors and highly purified, traceable pharmaceutical grade HSA is also a part of this grouping.

Although considerable progress has been made in the design of chemically defined xeno-free media, cell passage, and fold-expansion may be limited by the quality of culture supplements and their stability in culture. Daily replenishing of media is often recommended when using chemically defined media and this can be both costly and labor intensive.

Although chemically defined ECM based matrices have helped advance the design of xeno-free hESC culture models, they may still be limited in their ability to physiologically mimic the in vivo stem cell niche. Studies indicate distinct differences in cell signaling and behavior in cells grown in 2-D versus 3-D culture [105-107]. During embryogenesis, inner cell mass cells are embedded within a dynamic 3D stem cell niche, which helps direct both self-renewal and differentiation via the presentation of specific cell signaling molecules, regulation of matrix stiffness, and the establishment of cytokine gradients [108]. While the exact biomolecular composition of this native hESC environment has not been fully defined to date, it is likely that 2D systems will prove inadequate in functionally mimicking the in vivo microenvironment, ultimately restricting their ability to optimally support downstream differentiation applications. Bioengineered 3-D scaffolds offer an opportunity to modulate stem cell behavior in vitro to more closely resemble that occurring in vivo [109-112].

Numerous substrates for 3-D culture have been explored and tested. As a biomaterial, hydrogels are perhaps the most versatile and have been used in a variety of engineering applications. Hydrogels are a network of polymer chains either of natural or synthetic origin that are very absorbent and flexible due to high water content, thus resembling natural matrices. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) and a primary component of in vivo extracellular matrices, making it particularly attractive in designing 3-D scaffolding for stem cells. GAG’s are known participators in receptor signaling complexes, cell-cell adhesion, and cell-matrix interactions. HA-hydrogel architecture and rigidity can be easily manipulated. HA encapsulated hESCs were shown to retain pluripotency for 20 days in culture and the need for continuous stem cell passaging was eliminated [113]. The hydrogel scaffolds also allowed hESC to be driven along specific paths of differentiation. Stem cell release from the hydrogel necessitated enzymatic digestion with hyaluronidase which may however impact stem cell functionality in the long term. It should be noted MEF-conditioned medium was still used in this study.

Others have used a hydrogel scaffold composed of chitosan, a deceatylated cationic polysaccharide and alginate [114]. Investigators were able to show self-renewal and characteristic hESC gene activity for 21 days in the absence of feeder layers or conditioned medium. Direct implantation of hESC-scaffold constructs in to mice resulted in teratoma formation, with immunohistochemical evidence of all three germ layers. Microfiber constructs of chitosan-alginate also support hESC self-renewal under chemically defined conditions [115]. The 3-D microfiber culture model has the added benefit of allowing direct cryopreservation in situ of encapsulated cells. To date there has only been one report of hESC culture for more than 30 days in a 3-D matrix. Using alginate hydrogels, Siti-Ismail and colleagues [116] were able to maintain hESCs in a pluripotent state for 260 days, without enzymatic treatment and repeated passaging of the cells. This was accomplished using a basic stem cell maintenance medium DMEM with 20% knockout serum replacer and bFGF supplementation.

A plant-derived nanofibrillar cellulose (NFC) has similarly been used to create a flexible 3D environment [117]. This hydrogel system utilized maintained stem cells in culture as 3-D spheroids, under xeno-free and feeder-free conditions in a defined medium, TESR. Passaging was accomplished by enzymatically denaturing the matrix and transferring the cells to new NFC platforms every 7–12 days. The hESC could be maintained undifferentiated as 3-D spheroids for 26 days after which teratoma formation was observed. The undifferentiated hESCs were karyotypically normal and expressed mRNA for all pluripotency markers.

Synthetic polymers are especially valuable for 3-D scaffolding, offering the advantage of versatility. Microfabrication techniques can be used to generate scaffolds with specific chemical signals immobilized on the matrix. As discussed earlier, combining matrices with tissue-specific bioactive molecules such as cell-adhesion proteins, growth factors and peptides are particularly promising. Derivatives of polyethylene glycol (PEG) combined with tissue-specific bioactive molecules such as cell-adhesion proteins, growth factors and peptides are particularly promising. The presentation of physiologically relevant signals allows the hESC microenvironment to be tailored towards self-renewal or differentiation as needed. PEG hydrogel constructs cross-linked with matrix metalloproteinase (MMP)-sensitive peptides and coupled with integrin-specific adhesion ligands are able to sustain the pluripotency of mouse and human stem cells [118,119]. The 3-D PEG-based scaffolds supported the expansion, self-renewal and differentiation potential of pluripotent hESC lines H1, H9 and Novo, under feeder-free conditions in culture medium supplemented with bFGF and knock-out serum replacement. Increased mRNA expression of stemness- related genes were detected in hESCs after 3-D culture on PEG scaffolds as compared to 2-D culture on MEF feeders or in Matrigel.

Suspension culture of hESCs on microcarriers (MC) has also been used to create a pseudo 3D microenvironment that can be easily manipulated to direct cells towards undifferentiated proliferation or directed cell differentiation [94,104,120-127]. Microcarriers allow easy scale up in bioreactors to increase total stem cell production. Cell adhesion to microcarriers can be increased by altering their surface. hESC could be cultured on Matrigel coated cellulose beads in serum-free mTeSR and Stem Pro media for 20 passages without loss of stem cell characteristics [125]. Marinho and colleagues [123] developed a serum-free xeno-free culture system for hESC culture using polystyrene microcarrier spheres positively charged with triethyl-ammonium groups (Hillex II) and a defined medium BRASTEM supplemented with bFGF. High growth rate of stem cells and retention of pluripotency was demonstrated after six days in culture. Although promising, serial passaging of pluripotent hESCs on microcarriers over a much longer time interval are necessary to validate this xeno-free culture model for any clinical application. One of the problems with MC based suspension culture has been the tendency of hESC cells to aggregate resulting in multi-bead clusters with low speed agitation in the bioreactors. High levels of agitation resulted in shear stress which was also detrimental to continued propagation hESC cultures.

Suspension culture of hESCs without the use of coatings or microcarriers is also possible [128-131]. Aggregate cell suspension culture of hESCs has been shown to be a particularly favorable culture system for embryoid body formation, eliminating the need for extraneous scaffolds [130]. Steiner and colleagues devised a culture model for generating hESC lines from clusters of inner cell mass cells kept in suspension culture. The initial ICM were extracted from embryos using a laser. They were able to generate 3 new hESC lines under xeno-free conditions in defined medium supplemented with laminin, fibronectin, bFGF, Activin A and neurotrophins BDNF, NT3 and NT4. The new stem cell lines retained pluripotent characteristics even after 10 weeks in culture, were capable of forming all 3 germ layers and could undergo directed differentiation in to neural spheres. Loss of cell viability during passaging can however result in lower expansion rates in suspension culture as compared to that typically observed with adherent cell culture models. Wang et al. developed a scalable xeno-free and clinically compliant culture system for hiPSC suitable for both suspension and adherent culture [85]. This was achieved in the chemically defined medium, E8 that consists of just 8 components. hiPSC maintained pluripotency in culture expressing all stem cell markers and exhibiting karyotype stability after more than 30 passages. The induced pluripotent cells could be directed towards differentiation into hematopoetic cells and showed teratoma formation upon injection in to mice. Demonstrated efficacy combined with its simplicity make this model especially attractive and cost-effective for large scale production of hiPSCs. A 3-D sphere culture system using a methylcellulose polymer to suppress fusion of aggregates was recently described and may help to further optimize the suspension culture model [131]. Investigators tested the system with both mTESR1 and StemPro medium, with similar results as regards retention of pluripotency.