A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells.

Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Dopamine transporter neuroimaging accurately assesses the maturation of dopamine neurons in a preclinical model of Parkinson's disease.

BACKGROUND: Significant developments in stem cell therapy for Parkinson's disease (PD) have already been achieved; however, methods for reliable assessment of dopamine neuron maturation in vivo are lacking. Establishing the efficacy of new cellular therapies using non-invasive methodologies will be critical for future regulatory approval and application. The current study examines the utility of neuroimaging to characterise the in vivo maturation, innervation and functional dopamine release of transplanted human embryonic stem cell-derived midbrain dopaminergic neurons (hESC-mDAs) in a preclinical model of PD. METHODS: Female NIH RNu rats received a unilateral stereotaxic injection of 6-OHDA into the left medial forebrain bundle to create the PD lesion. hESC-mDA cell and sham transplantations were carried out 1 month post-lesion, with treated animals receiving approximately 4 × 105 cells per transplantation. Behavioural analysis, [18F]FBCTT and [18F]fallypride microPET/CT, was conducted at 1, 3 and 6 months post-transplantation and compared with histological characterisation at 6 months. RESULTS: PET imaging revealed transplant survival and maturation into functional dopaminergic neurons. [18F]FBCTT-PET/CT dopamine transporter (DAT) imaging demonstrated pre-synaptic restoration and [18F]fallypride-PET/CT indicated functional dopamine release, whilst amphetamine-induced rotation showed significant behavioural recovery. Moreover, histology revealed that the grafted cells matured differently in vivo producing high- and low-tyrosine hydroxylase (TH) expressing cohorts, and only [18F]FBCTT uptake was well correlated with differentiation. CONCLUSIONS: This study provides further evidence for the value of in vivo functional imaging for the assessment of cell therapies and highlights the utility of DAT imaging for the determination of early post-transplant cell maturation and differentiation of hESC-mDAs.

Influencing the Fate of Cardiac and Neural Stem Cell Differentiation Using Small Molecule Inhibitors of ALK5.

In this study, 50 tri-substituted imidazoles (TIs), which are analogs of the small molecules TA-01 and SB203580, were synthesized and screened for cardiomyogenic activities. Several TIs displayed cardiomyogenic activities when applied during the differentiation from days 3-5. The TIs did not affect the Wnt/ß-catenin pathway during cardiomyogenesis and the likely mechanism of action is through the inhibition of ALK5 of the TGFß pathway. Interestingly, these TIs promoted the neural differentiation of human pluripotent stem cells (hPSCs) with a similar potency to that of the dual SMAD inhibitors SB431542/LDN-193189 when dosed from days 1 to 9. The neural induction activities of the TIs correlated with their ALK5 inhibitory activities. This study reports the discovery of small molecule inhibitors of ALK5, which can promote the differentiation of hPSCs into cardiomyocytes or neural cells depending on the time of dosing, showing potential for the production of clinical-grade cardiac/neural cells for regenerative therapy. Stem Cells Translational Medicine 2018;7:709-720.

Immobilization of vitronectin-binding heparan sulfates onto surfaces to support human pluripotent stem cells.

Functionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence-dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence-based affinity chromatography could be used to isolate a VN-binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)-based assays. Plasma polymerization of allylamine (AA) to tissue culture-treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9-coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6-O- and N-sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS-coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity-purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887-1896, 2018.

Immature Midbrain Dopaminergic Neurons Derived from Floor-Plate Method Improve Cell Transplantation Therapy Efficacy for Parkinson's Disease.

Recent reports have indicated human embryonic stem cells-derived midbrain dopamine (mDA) neurons as proper cell resources for use in Parkinson's disease (PD) therapy. Nevertheless, no detailed and systematic study has been conducted to identify which differentiation stages of mDA cells are most suitable for transplantation in PD therapy. Here, we transplanted three types of mDA cells, DA progenitors (differentiated in vitro for 16 days [D16]), immature DA neurons (D25), and DA neurons (D35), into PD mice and found that all three types of cells showed high viability and strong neuronal differentiation in vivo. Both D25 and D35 cells showed neuronal maturation and differentiation toward TH+ cells and, accordingly, satisfactory behavioral functional recovery. However, transplanted D16 cells were less capable of producing functional recovery. These findings provide a valuable guideline for standardizing the differentiation stage of the transplantable cells used in clinical cell therapy for PD. Stem Cells Translational Medicine 2017;6:1803-1814.

Microcarrier-Expanded Neural Progenitor Cells Can Survive, Differentiate, and Innervate Host Neurons Better When Transplanted as Aggregates.

Neuronal progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) are an excellent cell source for transplantation therapy due to their availability and ethical acceptability. However, the traditional method of expansion and differentiation of hESCs into NPCs in monolayer cultures requires a long time, and the cell yield is low. A microcarrier (MC) platform can improve the expansion of hESCs and increase the yield of NPCs. In this study, for the first time, we transplanted microcarrier-expanded hESC-derived NPCs into the striatum of adult NOD-SCID IL2Rgc null mice, either as single cells or as cell aggregates. The recipient mice were perfused, and the in vivo survival, differentiation, and targeted innervation of the transplanted cells were assessed by immunostaining. We found that both the transplanted single NPCs and aggregate NPCs were able to survive 1 month posttransplantation, as revealed by human-specific neural cell adhesion molecule (NCAM) and human nuclear antigen staining. Compared to the single cells, the transplanted cell aggregates showed better survival over a 3-month period. In addition, both the transplanted single NPCs and the aggregate NPCs were able to differentiate into DCX-positive immature neurons and Tuj1-positive neurons in vivo by 1 month posttransplantation. However, only the transplantation of aggregate NPCs was shown to result in mature neurons at 3 months posttransplantation. Furthermore, we found that the cell aggregates were able to send long axons to innervate their targets. Our study provides preclinical evidence that the use of MCs to expand and differentiate hESC-derived NPCs and transplantation of these cells as aggregates produce longer survival in vivo.

Pushing the envelope in tissue engineering: ex vivo production of thick vascularized cardiac extracellular matrix constructs.

Functional vascularization is a prerequisite for cardiac tissue engineering of constructs with physiological thicknesses. We previously reported the successful preservation of main vascular conduits in isolated thick acellular porcine cardiac ventricular ECM (pcECM). We now unveil this scaffold's potential in supporting human cardiomyocytes and promoting new blood vessel development ex vivo, providing long-term cell support in the construct bulk. A custom-designed perfusion bioreactor was developed to remodel such vascularization ex vivo, demonstrating, for the first time, functional angiogenesis in vitro with various stages of vessel maturation supporting up to 1.7 mm thick constructs. A robust methodology was developed to assess the pcECM maximal cell capacity, which resembled the human heart cell density. Taken together these results demonstrate feasibility of producing physiological-like constructs such as the thick pcECM suggested here as a prospective treatment for end-stage heart failure. Methodologies reported herein may also benefit other tissues, offering a valuable in vitro setting for "thick-tissue" engineering strategies toward large animal in vivo studies.

Cardiomyocyte differentiation of pluripotent stem cells with SB203580 analogues correlates with Wnt pathway CK1 inhibition independent of p38 MAPK signaling.

Differentiation of human pluripotent stem cells as embryoid bodies (EBs) has been achieved previously with p38alfa MAPK inhibitors such as SB203580 with moderate efficiency of 10-15%. We synthesized and screened 42 compounds that are 2,4,5-trisubstituted azole analogues of SB203580 for efficient cardiomyocyte differentiation. Our screen identified novel compounds that have similar cardiac differentiation activity as SB203580. However, the cardiac differentiation did not correlate with p38alfa MAPK inhibition, indicating an alternative mechanism in cardiac differentiation. Upon profiling several 2,4,5-trisubstituted azole compounds against a panel of 97 kinases we identified several off targets, among them casein kinases 1 (CK1). The cardiomyogenic activities of SB203580 and its analogues showed a correlation with post mesoderm Wnt/beta-catenin pathway inhibition of CK1 epsilon and delta. These findings united the mechanism of 2,4,5-trisubstituted azole with the current theory of Wnt/beta-catenin regulated pathway of cardiac differentiation. Consequently an efficient cardiomyocyte protocol was developed with Wnt activator CHIR99021 and 2,4,5-trisubstituted azoles to give high yields of 50-70% cardiomyocytes and a 2-fold increase in growth.

High-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias.

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident with human pluripotent stem cells (hPSCs): microenvironmental heterogeneity limits hPSC cell fate control. We developed a high-throughput platform to screen hPSCs in configurable microenvironments in which we optimized colony size, cell density and other parameters to achieve rapid and robust cell fate responses to exogenous cues. We used this platform to perform single-cell protein expression profiling, revealing that Oct4 and Sox2 costaining discriminates pluripotent, neuroectoderm, primitive streak and extraembryonic cell fates. We applied this Oct4-Sox2 code to analyze dose responses of 27 developmental factors to obtain lineage-specific concentration optima and to quantify cell line-specific endogenous signaling pathway activation and differentiation bias. We demonstrated that short-term responses predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell lines reticent to cardiac induction. This platform will facilitate high-throughput hPSC-based screening and quantification of lineage-induction bias.

Electrical stimulation promotes maturation of cardiomyocytes derived from human embryonic stem cells.

While human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes, their immature phenotypes limit their therapeutic application for myocardial regeneration. We sought to determine whether electrical stimulation could enhance the differentiation and maturation of hESC-derived cardiomyocytes. Cardiac differentiation was induced in a HES3 hESC line via embryoid bodies formation treated with a p38 MAP kinase inhibitor. Detailed molecular and functional analysis were performed in those hESC-derived cardiomyocytes cultured for 4 days in the absence or presence of electrical field stimulation (6.6 V/cm, 1 Hz, and 2 ms pulses) using an eight-channel C-Pace stimulator (Ion-Optics Co., MA). Upon electrical stimulation, quantitative polymerase chain reaction demonstrated significant upregulation of cardiac-specific gene expression including HCN1, MLC2V, SCN5A, SERCA, Kv4.3, and GATA4; immunostaining and flow cytometry analysis revealed cellular elongation and an increased proportion of troponin-T positive cells (6.3 ± 1.2% vs. 15.8 ± 2.1%; n = 3, P < 0.01). Electrophysiological studies showed an increase in the proportion of ventricular-like hESC-derived cardiomyocytes (48 vs. 29%, P < 0.05) with lengthening of their action potential duration at 90% repolarization (387.7 ± 35.35; n = 11 vs. 291.8 ± 20.82; n = 10, P < 0.05) and 50% repolarization (313.9 ± 27.94; n = 11 vs. 234.0 ± 16.10; n = 10, P < 0.05) after electrical stimulation. Nonetheless, the membrane diastolic potentials and action potential upstrokes of different hESC-derived cardiomyocyte phenotypes, and the overall beating rate remained unchanged (all P > 0.05). Fluorescence confocal imaging revealed that electrical stimulation significantly increased both spontaneous and caffeine-induced calcium flux in the hESC-derived cardiomyocytes (approximately 1.6-fold for both cases; P < 0.01). In conclusion, electrical field stimulation increased the expression of cardiac-specific genes and the yield of differentiation, promoted ventricular-like phenotypes, and improved the calcium handling of hESC-derived cardiomyocytes.

3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].

BACKGROUND: Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS: hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. RESULTS: hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. CONCLUSION: This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system.

Microcarrier suspension cultures for high-density expansion and differentiation of human pluripotent stem cells to neural progenitor cells.

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×106 cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×106 cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into ßIII-tubulin⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.

Activated T cells modulate immunosuppression by embryonic-and bone marrow-derived mesenchymal stromal cells through a feedback mechanism.

BACKGROUND AIMS: Human embryonic stem cell (hESC)-derived mesenchymal stromal cells (MSC) (hESC-MSC) are an alternative source of MSC to bone marrow (BM)-derived MSC (BM-MSC), which are being investigated in clinical trials for their immunomodulatory potential. hESC-MSC have the advantage of being consistent because each batch can be generated from hESC under defined conditions. In contrast, BM-MSC have a limited proliferative capacity. METHODS: The ability to suppress the proliferation of anti-CD3/CD28-stimulated CD4 (+) T cells by hESC-MSC was compared with adult BM-MSC and neonatal foreskin fibroblast (Fb). RESULTS: hESC-MSC suppress the proliferation of CD4 (+) T cells in both contact and transwell systems, although inhibition is less in the transwell system. hESC-MSC are approximately 2-fold less potent (67 cells/100 T cells) than BM-MSC and Fb (37 and 34 cells/100 T cells, respectively) at suppressing T-cell proliferation by 50% in a transwell [inhibitory concentration(IC)(50)]. The anti-proliferative effect is not contact-dependent but requires the presence of factors such as interferon (IFN)-γ produced by activated T cells. IFN-γ induces the expression of indoleamine-2,3-dioxygenase (IDO) in hESC-MSC, BM-MSC and Fb, contributing to their immunosuppressive property. CONCLUSIONS: The feedback loop between MSC or Fb and activated T cells may limit the immunosuppressive effects of MSC and Fb to sites containing ongoing immunologic or inflammatory responses where activated T cells induce the up-regulation of IDO and immunomodulatory properties of MSC and Fb. These data demonstrate that hESC-MSC may be evaluated further as an allogeneic cell source for therapeutic applications requiring immunosuppression.

Agitation can induce differentiation of human pluripotent stem cells in microcarrier cultures.

One of the factors that can impact human embryonic stem cell expansion in stirred microcarrier culture reactors is mechanical stress caused by agitation. Therefore, we have investigated the effects of agitation on human embryonic stem cell growth and expression of pluripotent markers. Agitation of HES-2 cell line in microcarrier cultures in stirred spinner and agitated six-well plates did not affect expression of pluripotent markers, cell viability, and cell doubling times even after seven passages. However, HES-3 cell line was found to be shear sensitive, showing downregulation of three pluripotent markers Oct-4, mAb 84, and Tra-1-60, and lower cell densities in agitated as compared with static cultures, even after one passage. Cell viability was unaffected. The HES-3-agitated cultures showed increased expression of genes and proteins of the three germ layers. We were unable to prevent loss of pluripotent markers or restore doubling times in agitated HES-3 microcarrier cultures by addition of five different known cell protective polymers. In addition, the human induced pluripotent cell line IMR90 was also shown to differentiate in agitated conditions. These results indicate that the effect of agitation on cell growth and differentiation is cell line specific. We assume that the changes in the growth and differentiation of the agitation-sensitive (HES-3) cell line do not result from the effect of shear stress directly on cell viability, but rather by signaling effects that influence the cells to differentiate resulting in slower growth.

Defining a threshold surface density of vitronectin for the stable expansion of human embryonic stem cells.

Current methodology for pluripotent human embryonic stem cells (hESCs) expansion relies on murine sarcoma basement membrane substrates (Matrigel™), which precludes the use of these cells in regenerative medicine. To realize the clinical efficacy of hESCs and their derivatives, expansion of these cells in a defined system that is free of animal components is required. This study reports the successful propagation of hESCs (HES-3 and H1) for > 20 passages on tissue culture-treated polystyrene plates, coated from 5 µg/mL of human plasma-purified vitronectin (VN) solution. Cells maintain expression of pluripotent markers Tra1-60 and OCT-4 and are karyotypically normal after 20 passages of continuous culture. In vitro and in vivo differentiation of hESC by embryoid body formation and teratoma yielded cells from the ecto-, endo-, and mesoderm lineages. VN immobilized on tissue culture polystyrene was characterized using a combination of X-ray photoemission spectroscopy, atomic force microscopy, and quantification of the VN surface density with a Bradford protein assay. Ponceau S staining was used to measure VN adsorption and desorption kinetics. Tuning the VN surface density, via the concentration of depositing solution, revealed a threshold surface density of 250 ng/cm², which is required for hESCs attachment, proliferation, and differentiation. Cell attachment and proliferation assays on VN surface densities above this threshold show the substrate properties to be equally viable.

Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells.

The standard method for culturing human embryonic stem cells (hESC) uses supporting feeder layers of cells or an undefined substrate, Matrigel(™), which is a basement membrane extracted from murine sarcoma. For stem cell therapeutic applications, a superior alternative would be a defined, artificial surface that is based on immobilized human plasma vitronectin (VN), which is an adhesion-mediating protein. Therefore, VN adsorbed to diverse polymer surfaces was explored for the continuous propagation of hESC. Cells propagated on VN-coated tissue culture polystyrene (TCPS) are karyotypically normal after >10 passages of continuous culture, and are able to differentiate into embryoid bodies containing all three germ layers. Expansion rates and pluripotent marker expression verified that a minimal VN surface density threshold is required on TCPS. Further exploration of adsorbed VN was conducted on polymer substrates with different properties, ranging from hydrophilic to hydrophobic and including cationic and anionic polyelectrolyte coatings. Despite differing surface properties, these substrates adsorbed VN above the required surface density threshold and were capable of supporting hESC expansion for >10 passages. Correlating wettability of the VN-coated surfaces with the response of cultured hESC, higher cell expansion rates and OCT-4 expression levels were found for VN-coated TCPS, which exhibits a water contact angle close to 65°. Importantly, this simple, defined surface matches the performance of the benchmark Matrigel, which is a hydrogel with highly complex composition.

Expansion of human embryonic stem cells on cellulose microcarriers.

This unit describes the routine maintenance and expansion of undifferentiated human embryonic stem cells (hESC) on cellulose microcarriers. Conventionally, hESCs have been maintained on feeder cells or extracellular matrix-coated two-dimensional tissue culture plates. The expansion of hESC on a tissue culture platform is limited by the available surface area and the requirement of repetitive subculturing to reach the required cell yield. Here, we show that expansion of hESC can be carried out in a three-dimensional suspension culture using Matrigel-coated cellulose microcarriers. hESCs from a tissue culture plate can be seeded directly onto the microcarriers; hESC microcarrier culture is passaged and expanded by mechanical dissociation of the cells without enzyme. Expansion of the culture in a 100-ml spinner flask is also described. Long-term culture of hESC on the microcarriers maintains typical pluripotent markers (OCT-4, Tra-1-60, and SSEA-4) and stable karyotype. Spontaneous differentiations of microcarrier-maintained hESCs in vitro (embryoid body formation) and in vivo (teratoma formation in SCID mouse) have demonstrated formation of the three germ layers. These protocols can also be applied equally well to human induced pluripotent stem cells.

Establishing efficient siRNA knockdown in stem cells using fluorescent oligonucleotides.

The following protocols provide a rapid approach for establishing good working conditions for transfecting siRNAs for specific gene knockdown. By first using microscopy to evaluate efficient transfection of an inexpensive, fluorescent oligonucleotide, the researcher can later proceed with more expensive Western blot or quantitative real-time PCR (qRT-PCR) methods. Thus, the main culprit of ineffective knockdown, poor transfection, can be eliminated before engaging in tedious and time-consuming approaches for troubleshooting siRNA knockdown experiments.

Investigations into the metabolism of two-dimensional colony and suspended microcarrier cultures of human embryonic stem cells in serum-free media.

Metabolic studies of human embryonic stem cells (hESCs) can provide important information for stem cell bioprocessing. To this end, we have examined growth and metabolism of hESCs in both traditional 2-dimensional (2D) colony cultures and 3-dimensional microcarrier cultures using a conditioned medium and 3 serum-free media. The 2D colony cultures plateaued at cell densities of 1.1-1.5 × 106 cells/mL at day 6 due to surface limitation. Microcarrier cultures achieved 1.5-2 × 106 cells/mL on days 8-10 before reaching a plateau; this growth arrest was not due to surface limitation, but probably due to metabolic limitations. Metabolic analysis of the cultures showed that amino acids (including glutamine) and glucose are in excess and are not limiting cell growth; on the other hand, the high levels of waste products (25 mM lactate and 0.8 mM ammonium) and low pH (6.6) obtained at the last stages of cell propagation could be the causes for growth arrest. hESCs cultured in media supplemented with lactate (up to 28 mM) showed reduced cell growth, whereas ammonium (up to 5 mM) had no effect. Lactate and, to a lesser extent, ammonia affected pluripotency as reflected by the decreasing population of cells expressing pluripotent marker TRA-1-60. Feeding hESC cultures with low concentrations of glucose resulted in lower lactate levels (∼10%) and a higher pH level of 6.7, which leads to a 40% increase in cell density. We conclude that the high lactate levels and the low pH during the last stages of high-density hESC culture may limit cell growth and affect pluripotency. To overcome this limitation, a controlled feed of low levels of glucose and online control of pH can be used.