Spontaneous Single-Copy Loss of TP53 in Human Embryonic Stem Cells Markedly Increases Cell Proliferation and Survival.

Genomic aberrations have been identified in many human pluripotent stem cell (hPSC) cultures. Commonly observed duplications in portions of chromosomes 12p and 17q have been associated with increases in genetic instability and resistance to apoptosis, respectively. However, the phenotypic consequences related to sporadic mutations have not been evaluated to date. Here, we report on the effects of a single-copy deletion of the chr17p13.1 region, a sporadic mutation that spontaneously arose independently in several subclones of a human embryonic stem cell culture. Compared to cells with two normal copies of chr17p13.1 ("wild-type"), the cells with a single-copy deletion of this region ("mutant") displayed a selective advantage when exposed to stressful conditions, and retained a higher percentage of cells expressing the pluripotency marker POU5F1/OCT4 after 2 weeks of in vitro differentiation. Knockdown of TP53, which is a gene encompassed by the deleted region, in wild-type cells mimicked the chr17p13.1 deletion phenotype. Thus, sporadic mutations in hPSCs can have phenotypic effects that may impact their utility for clinical applications. Stem Cells 2017;35:872-885.

The tumorigenic potential of pluripotent stem cells: What can we do to minimize it?

Human pluripotent stem cells (hPSCs) have the potential to fundamentally change the way that we go about treating and understanding human disease. Despite this extraordinary potential, these cells also have an innate capability to form tumors in immunocompromised individuals when they are introduced in their pluripotent state. Although current therapeutic strategies involve transplantation of only differentiated hPSC derivatives, there is still a concern that transplanted cell populations could contain a small percentage of cells that are not fully differentiated. In addition, these cells have been frequently reported to acquire genetic alterations that, in some cases, are associated with certain types of human cancers. Here, we try to separate the panic from reality and rationally evaluate the true tumorigenic potential of these cells. We also discuss a recent study examining the effect of culture conditions on the genetic integrity of hPSCs. Finally, we present a set of sensible guidelines for minimizing the tumorigenic potential of hPSC-derived cells. © 2016 The Authors. Inside the Cell published by Wiley Periodicals, Inc.

Induced pluripotent stem cells from highly endangered species.

For some highly endangered species there are too few reproductively capable animals to maintain adequate genetic diversity, and extraordinary measures are necessary to prevent extinction. We report generation of induced pluripotent stem cells (iPSCs) from two endangered species: a primate, the drill, Mandrillus leucophaeus and the nearly extinct northern white rhinoceros, Ceratotherium simum cottoni. iPSCs may eventually facilitate reintroduction of genetic material into breeding populations.

Probing Multiple Transplant Delivery Routes of CD+34 Stem Cells for Promoting Behavioral and Histological Benefits in Experimental Ischemic Stroke.

Stroke is a leading cause of death in the US and around the world but with limited treatment options. Survivors often present with long-term cognitive and neurological deficits. Stem cell-based therapy has emerged as a potential treatment for stroke. While stem cell transplantation in stroke has reached clinical trials, mostly safety outcomes have been reported with efficacy readouts warranting more studies. In an effort to optimize the stem cell regimen for stroke, here we conducted vis-a-vis comparison of different routes of transplantation, namely, intracerebral, intraarterial, and intranasal delivery of expanded human CD34 + stem cells, called ProtheraCytes, in the established stroke model of transient middle cerebral artery occlusion (MCAO) using adult Sprague-Dawley rats. After adjusting for the dose and subacute timing of cell delivery, animals were randomly assigned to receive either ProtheraCytes or vehicle. Motor and neurological assays from days 7 to 28 post-stroke revealed significant functional recovery across all 3 delivery routes of ProtheraCytes compared to vehicle-treated stroke rats. Additionally, ProtheraCytes-transplanted stroke rats displayed significantly reduced infarct size and cell loss in the peri-infarct area coupled with enhanced neurogenesis and angiogenesis compared to vehicle-treated stroke rats. These results highlight the safety and efficacy of transplanting ProtheraCytes, including via the minimally invasive intranasal route, in conferring robust and stable behavioral and histological positive outcomes in experimental stroke.

Evaluation of expanded peripheral blood derived CD34+ cells for the treatment of moderate knee osteoarthritis.

Knee osteoarthritis (OA) is a degenerative joint disease of the knee that results from the progressive loss of articular cartilage. It is most common in the elderly and affects millions of people worldwide, leading to a continuous increase in the number of total knee replacement surgeries. These surgeries improve the patient's physical mobility, but can lead to late infection, loosening of the prosthesis, and persistent pain. We would like to investigate if cell-based therapies can avoid or delay such surgeries in patients with moderate OA by injecting expanded autologous peripheral blood derived CD34+ cells (ProtheraCytes®) into the articular joint. In this study we evaluated the survival of ProtheraCytes® when exposed to synovial fluid and their performance in vitro with a model consisting of their co-culture with human OA chondrocytes in separate layers of Transwells and in vivo with a murine model of OA. Here we show that ProtheraCytes® maintain high viability (>95%) when exposed for up to 96 hours to synovial fluid from OA patients. Additionally, when co-cultured with OA chondrocytes, ProtheraCytes® can modulate the expression of some chondrogenic (collagen II and Sox9) and inflammatory/degrading (IL1ß, TNF, and MMP-13) markers at gene or protein levels. Finally, ProtheraCytes® survive after injection into the knee of a collagenase-induced osteoarthritis mouse model, engrafting mainly in the synovial membrane, probably due to the fact that ProtheraCytes® express CD44, a receptor of hyaluronic acid, which is abundantly present in the synovial membrane. This report provides preliminary evidence of the therapeutic potential of CD34+ cells on OA chondrocytes in vitro and their survival after in vivo implantation in the knee of mice and merits further investigation in future preclinical studies in OA models.

Development of a potency assay for CD34+ cell-based therapy.

We have previously shown that intracardiac delivery of autologous CD34+ cells after acute myocardial infarction (AMI) is safe and leads to long term improvement. We are now conducting a multicenter, randomized, controlled Phase I/IIb study in post-AMI to investigate the safety and efficacy of intramyocardial injection of expanded autologous CD34+ cells (ProtheraCytes) (NCT02669810). Here, we conducted a series of in vitro studies characterizing the growth factor secretion, exosome secretion, gene expression, cell surface markers, differentiation potential, and angiogenic potential of ProtheraCytes clinical batches to develop a potency assay. We show that ProtheraCytes secrete vascular endothelial growth factor (VEGF) and its concentration is significantly correlated with the number of CD34+ cells obtained after expansion. ProtheraCytes also secrete exosomes containing proangiogenic miRNAs (126, 130a, 378, 26a), antiapoptotic miRNAs (21 and 146a), antifibrotic miRNAs (133a, 24, 29b, 132), and miRNAs promoting myocardial regeneration (199a and 590). We also show that ProtheraCytes have in vitro angiogenic activity, express surface markers of endothelial progenitor cells, and can differentiate in vitro into endothelial cells. After the in vitro characterization of multiple ProtheraCytes clinical batches, we established that measuring the concentration of VEGF provided the most practical, reliable, and consistent potency assay.

Kinetics of anti-SARS-CoV-2 IgG antibody levels and potential influential factors in subjects with COVID-19: A 11-month follow-up study.

We aim to study kinetics of anti-SARS-CoV-2 IgG antibody levels in subjects with COVID-19 for up to 11 months and the potential influential factors. The study was a prospective longitudinal study. The analyses were based on 77 serum/plasma samples with a mean of 4 samples per participant (range 1 - 18) in 20 participants with at least one positive Polymerase Chain Reaction testing result from 19 March 2020 up to 10 February 2021. Among the subjects (median age 34.5 years, 65% male), IgG level declined with the follow-up time (per month; geometric mean ratio [GMR] 0.73; 95% CI, 0.72 - 0.74). In a small sample of subjects from the general population with COVID-19, IgG levels declined non-linearly from month 2 to 11 with individual heterogeneity in quantity and changing speed and may be associated with gender, race and the loss of smell and taste.

Antagonism of human adiponectin receptors and their membrane progesterone receptor paralogs by TNFalpha and a ceramidase inhibitor.

The progestin and AdipoQ receptor (PAQR) family of proteins comprises three distinct structural classes, each with seemingly different agonist specificities. For example, Class I receptors, like the human adiponectin receptors (AdipoR1 and AdipoR2), sense proteins with a particular three-dimensional fold, while Class II receptors are nonclassical membrane receptors for the steroid hormone progesterone. Using a previously developed heterologous expression system to study PAQR receptor activity, we demonstrate that human PAQRs from all three classes are antagonized by both 1(S),2(R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, a ceramidase inhibitor, and TNFalpha, a homologue of adiponectin that functions antagonistically to both adiponectin and progesterone in human cells.

Sphingolipids function as downstream effectors of a fungal PAQR.

The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as d-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steady-state levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.

Adiponectin identified as an agonist for PAQR3/RKTG using a yeast-based assay system.

The PAQR family of proteins comprises an intriguing group of newly discovered receptors. Although the agonist is known for 5 of the 11 human PAQRs, most are considered "orphan" receptors. We developed a yeast-based assay system for PAQR receptor activity that can be used to identify agonists for PAQRs of unknown function. Using this system, we found that the proteinaceous hormone adiponectin functions as an agonist of PAQR3, a previously uncharacterized member of this family. This is not surprising given that PAQR3 is most closely related to PAQR1 (AdipoR1) and PAQR2 (AdipoR2), which also sense adiponectin. The identification of adiponectin as an agonist for PAQR3 is of considerable clinical relevance because adiponectin suppresses the proliferation of tumor cells and it has been reported that PAQR3 suppresses tumorigenesis. Thus, the interaction between PAQR3 and adiponectin may help explain the antiproliferative properties of adiponectin.

Derivation of Neural Stem Cells from Human Parthenogenetic Stem Cells.

We have previously shown that human parthenogenetic stem cells (hpSC) can be chemically directed to differentiate into a homogeneous population of multipotent neural stem cells (hpNSC) that are scalable, cryopreservable, express all the appropriate neural markers, and can be further differentiated into functional dopaminergic neurons. Differentiation of hpSC into hpNSC provides a platform to study the molecular basis of human neural differentiation, to develop cell culture models of neural disease, and to provide neural stem cells for the treatment of neurodegenerative diseases. Additionally, the hpNSC that are generated could serve as a platform for drug discovery and the determination of pharmaceutical-induced neural toxicity. Here, we describe in detail the stepwise protocol that was developed in our laboratory that facilitates the highly efficient and reproducible differentiation of hpSC into hpNSC.

Human parthenogenetic neural stem cell grafts promote multiple regenerative processes in a traumatic brain injury model.

International Stem Cell Corporation human parthenogenetic neural stem cells (ISC-hpNSC) have potential therapeutic value for patients suffering from traumatic brain injury (TBI). Here, we demonstrate the behavioral and histological effects of transplanting ISC-hpNSC intracerebrally in an animal model of TBI. Methods: Sprague-Dawley rats underwent a moderate controlled cortical impact TBI surgery. Transplantation occurred at 72 h post-TBI with functional readouts of behavioral and histological deficits conducted during the subsequent 3-month period after TBI. We characterized locomotor, neurological, and cognitive performance at baseline (before TBI), then on days 0, 1, 7, 14, 30, 60, and 90 (locomotor and neurological), and on days 28-30, 58-60, and 88-90 (cognitive) after TBI. Following completion of behavioral testing at 3 months post-TBI, animals were euthanized by transcardial perfusion and brains harvested to histologically characterize the extent of brain damage. Neuronal survival was revealed by Nissl staining, and stem cell engraftment and host tissue repair mechanisms such as the anti-inflammatory response in peri-TBI lesion areas were examined by immunohistochemical analyses. Results: We observed that TBI groups given high and moderate doses of ISC-hpNSC had an improved swing bias on an elevated body swing test for motor function, increased scores on forelimb akinesia and paw grasp neurological tests, and committed significantly fewer errors on a radial arm water maze test for cognition. Furthermore, histological analyses indicated that high and moderate doses of stem cells increased the expression of phenotypic markers related to the neural lineage and myelination and decreased reactive gliosis and inflammation in the brain, increased neuronal survival in the peri-impact area of the cortex, and decreased inflammation in the spleen at 90 days post-TBI. Conclusion: These results provide evidence that high and moderate doses of ISC-hpNSC ameliorate TBI-associated histological alterations and motor, neurological, and cognitive deficits.

Probing the mechanism of FET3 repression by Izh2p overexpression.

We previously reported a role for the IZH2 gene product in metal ion metabolism. Subsequently, Izh2p was also identified as a member of the PAQR family of receptors and, more specifically, as the receptor for the plant protein osmotin. In this report, we investigate the effect of Izh2p on iron homeostasis. We show that overproduction of Izh2p prevents the iron-dependent induction of the Fet3p component of the high-affinity iron-uptake system and is deleterious for growth in iron-limited medium. We demonstrate that the effect of Izh2p requires cAMP-dependent kinase and AMP-dependent kinase and is not mediated by general inhibition of the Aft1p iron-responsive transcriptional activator. We also show that Izh2p-overproduction negatively regulates Nrg1p/Nrg2p- and Msn2p/Msn4p-dependent reporters. Furthermore, we show that the Nrg1p/Nrg2p and Msn2p/Msn4p pairs are epistatic to each other with respect to their effects on FET3 expression. Finally, we show that the mechanism by which PAQR receptors activate signal transduction pathways is likely to be conserved from yeast to humans.

Heterologous expression of human mPRalpha, mPRbeta and mPRgamma in yeast confirms their ability to function as membrane progesterone receptors.

The nuclear progesterone receptor (nPR) mediates many of the physiological effects of progesterone by regulating the expression of genes, however, progesterone also exerts non-transcriptional (non-genomic) effects that have been proposed to rely on a receptor that is distinct from nPR. Several members of the progestin and AdipoQ-Receptor (PAQR) family were recently identified as potential mediators of these non-genomic effects. Membranes from cells expressing these proteins, called mPRalpha, mPRbeta and mPRgamma, were shown to specifically bind progesterone and have G-protein coupled receptor (GPCR) characteristics, although other studies dispute these findings. To clarify the role of these mPRs in non-genomic progesterone signaling, we established an assay for PAQR functional evaluation using heterologous expression in Saccharomyces cerevisiae. Using this assay, we demonstrate unequivocally that mPRalpha, mPRbeta and mPRgamma can sense and respond to progesterone with EC(50) values that are physiologically relevant. Agonist profiles also show that mPRalpha, mPRbeta and mPRgamma are activated by ligands, such as 17alpha-hydroxyprogesterone, that are known to activate non-genomic pathways but not nPR. These results strongly suggest that these receptors may indeed function as the long-sought-after membrane progesterone receptors. Additionally, we show that two uncharacterized PAQRs, PAQR6 and PAQR9, are also capable of responding to progesterone. These mPR-like PAQRs have been renamed as mPRdelta (PAQR6) and mPRvarepsilon (PAQR9). Additional characterization of mPRgamma and mPRalpha indicates that their progesterone-dependent signaling in yeast does not require heterotrimeric G-proteins, thus calling into question the characterization of the mPRs as a novel class of G-protein coupled receptor.

Novel Approach to Stem Cell Therapy in Parkinson's Disease.

In this commentary we discuss International Stem Cell Corporation's (ISCO's) approach to developing a pluripotent stem cell based treatment for Parkinson's disease (PD). In 2016, ISCO received approval to conduct the world's first clinical study of a pluripotent stem cell based therapy for PD. The Australian regulatory agency Therapeutic Goods Administration (TGA) and the Melbourne Health's Human Research Ethics Committee (HREC) independently reviewed ISCO's extensive preclinical data and granted approval for the evaluation of a novel human parthenogenetic derived neural stem cell (NSC) line, ISC-hpNSC, in a PD phase 1 clinical trial ( ClinicalTrials.gov NCT02452723). This is a single-center, open label, dose escalating 12-month study with a 5-year follow-up evaluating a number of objective and patient-reported safety and efficacy measures. A total of 6 years of safety and efficacy data will be collected from each patient. Twelve participants are recruited in this study with four participants per single dose cohort of 30, 50, and 70 million ISC-hpNSC. The grafts are placed bilaterally in the caudate nucleus, putamen, and substantia nigra by magnetic resonance imaging-guided stereotactic surgery. Participants are 30-70 years old with idiopathic PD ≤13 years duration and unified PD rating scale motor score (Part III) in the "OFF" state ≤49. This trial is fully funded by ISCO with no economic involvement from the patients. It is worth noting that ISCO underwent an exhaustive review process and successfully answered the very comprehensive, detailed, and specific questions posed by the TGA and HREC. The regulatory/ethic review process is based on applying scientific and clinical expertise to decision-making, to ensure that the benefits to consumers outweigh any risks associated with the use of medicines or novel therapies.

Supplementation of specific carbohydrates results in enhanced deposition of chondrogenic-specific matrix during mesenchymal stem cell differentiation.

Repair or regeneration of hyaline cartilage in knees, shoulders, intervertebral discs, and other assorted joints is a major therapeutic target. To date, therapeutic strategies utilizing chondrocytes or mesenchymal stem cells are limited by expandability or the generation of mechanically inferior cartilage. Our objective is to generate robust cartilage-specific matrix from human mesenchymal stem cells suitable for further therapeutic development. Human mesenchymal stem cells, in an alginate 3D format, were supplied with individual sugars and chains which comprise the glycan component of proteoglycans in articular cartilage (galactose, hyaluronic acid, glucuronic acid, and xylose) during chondrogenesis. After an initial evaluation for proteoglycan deposition utilizing Alcian blue, the tissue was further evaluated for viability, structural elements, and hypertrophic status. With the further addition of serum, a substantial increase was observed in viability, the amount of proteoglycan deposition, glycosaminoglycan production, and an enhancement of Hyaluronic Acid, Collagen II and Aggrecan deposition. Suppression of hypertrophic markers (COL1A1, COL10A1, MMP13, and RUNX2) was also observed. When mesenchymal stem cells were supplied with the raw building materials of proteoglycans and a limited amount of serum during chondrogenesis, it resulted in the generation of viable hyaline-like cartilage with deposition of structural components which exceeded previously reported in vitro-based cartilage.

Neural Stem Cell Tumorigenicity and Biodistribution Assessment for Phase I Clinical Trial in Parkinson's Disease.

Human pluripotent stem cells (PSC) have the potential to revolutionize regenerative medicine. However undifferentiated PSC can form tumors and strict quality control measures and safety studies must be conducted before clinical translation. Here we describe preclinical tumorigenicity and biodistribution safety studies that were required by the US Food and Drug Administration (FDA) and Australian Therapeutic Goods Administration (TGA) prior to conducting a Phase I clinical trial evaluating the safety and tolerability of human parthenogenetic stem cell derived neural stem cells ISC-hpNSC for treating Parkinson's disease (ClinicalTrials.gov Identifier NCT02452723). To mitigate the risk of having residual PSC in the final ISC-hpNSC population, we conducted sensitive in vitro assays using flow cytometry and qRT-PCR analyses and in vivo assays to determine acute toxicity, tumorigenicity and biodistribution. The results from these safety studies show the lack of residual undifferentiated PSC, negligible tumorigenic potential by ISC-hpNSC and provide additional assurance to their clinical application.

Neural Stem Cells Derived from Human Parthenogenetic Stem Cells Engraft and Promote Recovery in a Nonhuman Primate Model of Parkinson's Disease.

Cell therapy has attracted considerable interest as a promising therapeutic alternative for patients with Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying potentially viable human embryos and can be used to generate an unlimited supply of neural cells for transplantation. We have previously reported that human parthenogenetic stem cell-derived neural stem cells (hpNSCs) successfully engraft, survive long term, and increase brain dopamine (DA) levels in rodent and nonhuman primate models of PD. Here we report the results of a 12-month transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical parkinsonian symptoms. The hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immunosuppressed monkeys. Transplantation of hpNSCs was safe and well tolerated by the animals with no dyskinesia, tumors, ectopic tissue formation, or other test article-related serious adverse events. We observed that hpNSCs promoted behavioral recovery; increased striatal DA concentration, fiber innervation, and number of dopaminergic neurons; and induced the expression of genes and pathways downregulated in PD compared to vehicle control animals. These results provide further evidence for the clinical translation of hpNSCs and support the approval of the world's first pluripotent stem cell-based phase I/IIa study for the treatment of PD (Clinical Trial Identifier NCT02452723).

Proof of concept studies exploring the safety and functional activity of human parthenogenetic-derived neural stem cells for the treatment of Parkinson's disease.

Recent studies indicate that human pluripotent stem cell (PSC)-based therapies hold great promise in Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. Here we evaluate for the first time the safety and engraftment of human parthenogenetic stem cell-derived neural stem cells (hpNSCs) in two animal models: 6-hydroxydopamine (6-OHDA)-lesioned rodents and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates (NHPs). In both rodents and nonhuman primates, we observed successful engraftment and higher dopamine levels in hpNSC-transplanted animals compared to vehicle control animals, without any adverse events. These results indicate that hpNSCs are safe, well tolerated, and could potentially be a source for cell-based therapies in PD.

Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.