Encapsulation of Human-Bone-Marrow-Derived Mesenchymal Stem Cells in Small Alginate Beads Using One-Step Emulsification by Internal Gelation: In Vitro, and In Vivo Evaluation in Degenerate Intervertebral Disc Model.

Cell microencapsulation in gel beads contributes to many biomedical processes and pharmaceutical applications. Small beads (<300 µm) offer distinct advantages, mainly due to improved mass transfer and mechanical strength. Here, we describe, for the first time, the encapsulation of human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in small-sized microspheres, using one-step emulsification by internal gelation. Small (127−257 µm) high-mannuronic-alginate microspheres were prepared at high agitation rates (800−1000 rpm), enabling control over the bead size and shape. The average viability of encapsulated hBM-MSCs after 2 weeks was 81 ± 4.3% for the higher agitation rates. hBM-MSC-loaded microspheres seeded within a glycosaminoglycan (GAG) analogue, which was previously proposed as a mechanically equivalent implant for degenerate discs, kept their viability, sphericity, and integrity for at least 6 weeks. A preliminary in vivo study of hBM-MSC-loaded microspheres implanted (via a GAG-analogue hydrogel) in a rat injured intervertebral disc model demonstrated long-lasting viability and biocompatibility for at least 8 weeks post-implantation. The proposed method offers an effective and reproducible way to maintain long-lasting viability in vitro and in vivo. This approach not only utilizes the benefits of a simple, mild, and scalable method, but also allows for the easy control of the bead size and shape by the agitation rate, which, overall, makes it a very attractive platform for regenerative-medicine applications.

Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells.

Embryonic stem cell (ESC) self-renewal and cell fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during early stem cell differentiation. Notably, many RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly targeted by this factor. Using cross-linking and immunoprecipitation (CLIP-seq), we find that the pluripotency-associated STAT3 and OCT4 transcription factors interact with RNA in hESCs and confirm the binding of STAT3 to the conserved NORAD long-noncoding RNA. Our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated.

Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells.

Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.

Sources and derivation of human embryonic stem cells.

Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass (ICM) of the developing embryo. hESCs culture as cell lines in vitro and possess great potential in such research fields as developmental biology and cell-based therapy, as well as such industrial purposes as drug screening and toxicology. When ESCs were first derived by Thomson and colleagues, traditional methods of immunostaining and culturing, using primary mouse embryonic fibroblasts and medium supplemented by serum were used. Considerable efforts have since led to improved methods for isolating new lines in defined and reproducible conditions. This chapter discusses sources for embryos for ESC isolation, commonly used methods for deriving hESC lines, and a number of possible culture systems.

Screening of human pluripotent stem cells using CGH and FISH reveals low-grade mosaic aneuploidy and a recurrent amplification of chromosome 1q.

Pluripotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source for basic and applied research as well as in therapeutic medicine. The introduction of human induced pluripotent cells (hiPSCs) holds great promise for patient-tailored regenerative medicine therapies. However, for hESCs and hiPSCs to be applied for therapeutic purposes, long-term genomic stability in culture must be maintained. Until recently, G-banding analysis was considered as the default approach for detecting chromosomal abnormalities in stem cells. Our goal in this study was to apply fluorescence in-situ hybridization (FISH) and comparative genomic hybridization (CGH) for the screening of pluripotent stem cells, which will enable us identifying chromosomal abnormalities in stem cells genome with a better resolution. We studied three hESC lines and two hiPSC lines over long-term culture. Aneuploidy rates were evaluated at different passages, using FISH probes (12,13,16,17,18,21,X,Y). Genomic integrity was shown to be maintained at early passages of hESCs and hiPSCs but, at late passages, we observed low rates mosaiciam in hESCs, which implies a direct correlation between number of passages and increased aneuploidy rate. In addition, CGH analysis revealed a recurrent genomic instability, involving the gain of chromosome 1q. This finding was detected in two unrelated cell lines of different origin and implies that gains of chromosome 1q may endow a clonal advantage in culture. These findings, which could only partially be detected by conventional cytogenetic methods, emphasize the importance of using molecular cytogenetic methods for tracking genomic instability in stem cells.

Efficient engineering of vascularized ectopic bone from human embryonic stem cell-derived mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) can be derived from various adult and fetal tissues. However, the quality of tissues for the isolation of adult and fetal hMSCs is donor dependent with a nonreproducible yield. In addition, tissue engineering and cell therapy require large-scale production of a pure population of lineage-restricted stem cells that can be easily induced to differentiate into a specific cell type. Therefore, human embryonic stem cells (hESCs) can provide an alternative, plentiful source for generation of reproducible hMSCs. We have developed efficient differentiation protocols for derivation of hMSCs from hESCs, including coculture with murine OP9 stromal cells and feeder layer-free system. Our protocols have resulted in the generation of up to 49% of hMSCs, which expressed CD105, CD90, CD29, and CD44. The hMSCs exhibited high adipogenic, chondrocytic, and osteogenic differentiation in vitro. The latter correlated with osteocalcin secretion and vascular endothelial growth factor (VEGF) production by the differentiating hMSCs. hMSC-derived osteoblasts further differentiated and formed ectopic bone in vivo, and induced the formation of blood vessels in Matrigel implants. Our protocol enables generation of a purified population of hESC-derived MSCs, with the potential of differentiating into several mesodermal lineages, and particularly into vasculogenesis-inducing osteoblasts, which can contribute to the development of bone repair protocols.

Multipotent vasculogenic pericytes from human pluripotent stem cells promote recovery of murine ischemic limb.

BACKGROUND: Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. METHODS AND RESULTS: We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105(+)CD90(+)CD73(+)CD31(-) multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor ß, but not the smooth muscle cell marker α-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. CONCLUSIONS: Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine.

Proteomics profiling of human embryonic stem cells in the early differentiation stage.

The regulatory pathways responsible for maintaining human embryonic stem cells (hESCs) in an undifferentiated state have yet to be elucidated. Since these pathways are thought to be governed by complex protein cues, deciphering the changes that occur in the proteomes of the ESCs during differentiation is important for understanding the expansion and differentiation processes involved. In this study, we present the first quantitative comparison of the hESC protein profile in the undifferentiated and early differentiated states. We used iTRAQ (isobaric tags for relative and absolute quantification) labeling combined with two dimensional capillary chromatography coupled with tandem mass spectrometry (µLC-MS/MS) to achieve comparative proteomics of hESCs at the undifferentiated stage, and at 6, 48, and 72 h after initiation of differentiation. In addition, two dimensional electrophoresis (2-DE) was performed on differentiating hESCs at eleven points of time during the first 72 h of differentiation. The results indicate that during the first 48 h of hESC differentiation, many processes are initiated and are later reversed, including chromatin remodeling, heterochromatin spreading, a decrease in transcription and translation, a decrease in glycolytic proteins and cytoskeleton remodeling, and a decrease in focal and cell adhesion. Only 72 h after differentiation induction did the expression of the homeobox prox1 protein increase, indicating the beginning of developmental processes.

Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells.

Human pluripotent (embryonic or induced) stem cells (hPSCs) have many potential applications, not only for research purposes but also for clinical and industrial uses. While culturing these cells as undifferentiated lines, an adherent cell culture based on supportive layers or matrices is most often used. However, the use of hPSCs for industrial or clinical applications requires a scalable, reproducible and controlled process. Here we present a suspension culture system for undifferentiated hPSCs, based on a serum-free medium supplemented with interleukins and basic fibroblast growth factor, suitable for the mass production of these cells. The described system supports a suspension culture of hPSC lines, in both static and dynamic cultures. Results showed that hPSCs cultured with the described dynamic method maintained all hPSC features after 20 passages, including stable karyotype and pluripotency, and increased in cell numbers by 25-fold in 10 d. Thus, the described suspension method is suitable for large-scale culture of undifferentiated hPSCs.

Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells.

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS-derived cardiomyocytes), in the present study we investigated in iPS-derived cardiomyocytes, the functional properties related to [Ca(2+) ](i) handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca(2+) release to contraction and the b-adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Our major findings showed that iPS-derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force-frequency relations and mild (compared to adult) post-rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR-Ca(2+) handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF-derived iPS, the functional properties related to excitation-contraction coupling, resemble in part those of adult cardiomyocytes.

Suspension culture of undifferentiated human embryonic and induced pluripotent stem cells.

Alongside their contribution to research, human embryonic stem cells (hESC) may also prove valuable for cell-based therapies. Traditionally, these cells have been grown in adhesion culture either with or without feeder cells, allowing for their continuous growth as undifferentiated cells. However, to be applicable in therapy and industry they must be produced in a scalable and controlled process. Here we present for the first time a suspension culture system for undifferentiated hESC and induced pluripotent stem cells (iPSC), based on medium supplemented with the IL6RIL6 chimera (interleukin-6 receptor fused to interleukin-6), and basic fibroblast growth factor. Four hESC lines cultured in this system maintained all ESC features after 20 passages, including stable karyotype and pluripotency. Similar results were obtained when hESC were replaced with iPSC from two different cell lines. We demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated hESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications.

Human embryonic stem cell-derived cardiomyocytes can mobilize 1,4,5-inositol trisphosphate-operated [Ca2+]i stores: the functionality of angiotensin-II/endothelin-1 signaling pathways.

Because previous findings showed that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the machinery for Ca2+-induced release of calcium is immature, we tested the hypothesis that hESC-CM contain functional 1,4,5-inositol triphosphate (IP3)-operated intracellular Ca2+ ([Ca2+]i) stores. We investigated the effects of angiotensin II (AT-II) and endothelin 1 (ET-1), which activate the 1,4,5-IP3 pathway, on [Ca2+]i transients and contractions in hESC-CM. Our major findings were that in hESC-CM, both AT-II (10(-9)-10(-7) M) and ET-1 (10(-9)-10(-7) M) exert inotropic and lusitropic effects. The involvement of 1,4,5-IP3-dependent intracellular Ca2+ release in AT-I-induced effects was supported by these findings: the effects of AT-II were blocked by 2-aminoethoxyphenyl borate (2-APB, a 1,4,5-IP3 receptor blocker) and U73122 (a phosopholipase C blocker); and hESC-CM express AT-II type 1 and IP3 type I and II receptors as determined by fluorescence immunostaining. In conclusion, hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP3-operated releasable Ca2+ stores.

Embryonic stem cells: isolation, characterization and culture.

Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

Genetic modification of human embryonic stem cells with adenoviral vectors: differences of infectability between lines and correlation of infectability with expression of the coxsackie and adenovirus receptor.

Adenovirus is an efficient vector for expression of transgenes in dividing and nondividing cells. However, very few studies of human embryonic stem cells (hESCs) have utilized adenoviral vectors. We examine here the ability of adenovirus to infect naive hESCs and the differentiated derivatives of multiple hESC lines. We found a striking variation in adenovirus infection rates between lines. The variability in infection rates was positively correlated with the expression of the coxsackievirus and adenovirus receptor, but not that of alpha(nu)-integrin. Adenoviral infection did not interfere with the expression of pluripotency markers, even after passaging. In addition, infection did not affect differentiation of hESC-derived neural precursors in vitro. We also found that green fluorescent protein expression mediated by adenovirus can be a useful marker for tracking hESC in xenografts. We conclude that adenovirus is a practical vector for genetic modification of naive hESC from most, but not all lines, but may be more generally useful for gene transfer into differentiated derivatives of hESC lines.

Establishing a dynamic process for the formation, propagation, and differentiation of human embryoid bodies.

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe here a robust and well-defined scale up platform for human embryoid body (EB) formation, propagation, and differentiation. The efficacy of the dynamic process as compared to the static cultivation in Petri dishes was analyzed. Our optimized conditions include specific bioreactor and impeller type, seeding and propagation parameters, and scale up. Quantitative analyses of viable cell concentrations, apoptosis percentages, and EB yield revealed 6.7-fold enhancement in the generation of hESC-derived cells after 10 cultivation days. Other metabolic indices such as glucose consumption, lactic acid production and pH all pointed to efficient cell expansion in the dynamic cultures. The hydrodynamic conditions during seeding and cultivation were found to be crucial for the EB formation and propagation. The EBs' prearrangement in the static system and EB cultivation in the Glass Ball Impeller spinner flask resulted in high EB yield, a round homogenous shape, and the fastest growth rate. The appearance of representative genes of the three germ layers as well as primitive neuronal tube organization and blood vessel formation indicated that the initial developmental events in the human EBs are not interfered by the dynamic system. Furthermore, well developed endothelial networks and contracting EBs with functional cardiac muscle were also obtained after two cultivation weeks. Collectively, our study defines the technological platform for the controlled large-scale generation of hESC-derived cells for clinical and industrial applications.

1,4,5-Inositol trisphosphate-operated intracellular Ca(2+) stores and angiotensin-II/endothelin-1 signaling pathway are functional in human embryonic stem cell-derived cardiomyocytes.

On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca(2+)-induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP(3))-operated intracellular Ca(2+) ([Ca(2+)](i)) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP(3) pathway, on [Ca(2+)](i) transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10(-9) to 10(-7) M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10(-9) to 10(-7) M) increased the [Ca(2+)](i) transient amplitude. The involvement of 1,4,5-IP(3)-dependent intracellular Ca(2+) release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP(3) receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 microM 2-aminoethoxyphenyl borate (a 1,4,5-IP(3) receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP(3)-operated releasable Ca(2+) stores.

X-inactivation reveals epigenetic anomalies in most hESC but identifies sublines that initiate as expected.

The clinical and research value of human embryonic stem cells (hESC) depends upon maintaining their epigenetically naïve, fully undifferentiated state. Inactivation of one X chromosome in each cell of mammalian female embryos is a paradigm for one of the earliest steps in cell specialization through formation of facultative heterochromatin. Mouse ES cells are derived from the inner cell mass (ICM) of blastocyst stage embryos prior to X-inactivation, and cultured murine ES cells initiate this process only upon differentiation. Less is known about human X-inactivation during early development. To identify a human ES cell model for X-inactivation and study differences in the epigenetic state of hESC lines, we investigated X-inactivation in all growth competent, karyotypically normal, NIH approved, female hESC lines and several sublines. In the vast majority of undifferentiated cultures of nine lines examined, essentially all cells exhibit hallmarks of X-inactivation. However, subcultures of any hESC line can vary in X-inactivation status, comprising distinct sublines. Importantly, we identified rare sublines that have not yet inactivated Xi and retain competence to undergo X-inactivation upon differentiation. Other sublines exhibit defects in counting or maintenance of XIST expression on Xi. The few hESC sublines identified that have not yet inactivated Xi may reflect the earlier epigenetic state of the human ICM and represent the most promising source of NIH hESC for study of human X-inactivation. The many epigenetic anomalies seen indicate that maintenance of fully unspecialized cells, which have not formed Xi facultative heterochromatin, is a delicate epigenetic balance difficult to maintain in culture.

A pure population of ectodermal cells derived from human embryonic stem cells.

Embryonic stem (ES) cells represent a unique cellular model to recapitulate in vitro early steps of embryonic development and an unlimited cellular source in therapy for many diseases, as well as targets for drug discovery and toxicology screens. Although previous studies have reported epidermal differentiation of mouse and human embryonic stem (huES) cells, the heterogeneity of the resulting cell culture impairs the evaluation of differentiated cells for cell therapy. We report here the reproducible isolation of a homogenous ectodermal cell population, IT1, from human ES cells. Like primary cells, IT1 cells remain homogenous over 15 passages, expand up to 60 population doublings, and then die through senescence. Accordingly, IT1 cells display a normal karyotype and a somatic cell cycle kinetics and do not produce teratoma in nude mice. The production of K14-expressing epithelial cells driven by p63 expression strengthens the ectodermal nature of IT1 cells. Since IT1 can be isolated from different huES cell lines, it may provide a ready source of ectodermal progenitors for the development of a toxicology cell model, new-drug-screening strategies, and cell therapy transplantation.

Functional and developmental properties of human embryonic stem cells-derived cardiomyocytes.

Cardiovascular diseases are the most frequent cause of death in the industrialized world, with the main contributor being myocardial infarction. Given the high morbidity and mortality rates associated with congestive heart failure, the shortage of donor hearts for transplantation, complications resulting from immunosuppression, and long-term failure of transplanted organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. Because it is desired that the transplanted cells fully integrate within the diseased myocardium, contribute to its contractile performance, and respond appropriately to various physiological stimuli (eg, beta-adrenergic stimulation), our major long-term goal is to investigate the developmental changes in functional properties and hormonal responsiveness of human embryonic stem cells-derived cardiomyocytes (hESC-CM). Furthermore, because one of the key obstacles in advancing cardiac cell therapy is the low differentiation rate of hESC into cardiomyocytes, which reduces the clinical efficacy of cell transplantation, our second major goal is to develop efficient protocols for directing the cardiomyogenic differentiation of hESC in vitro. To accomplish the first goal, we investigated the functional properties of hESC-CM (<90 days old), respecting the contractile function and the underlying intracellular Ca(2+) handling. In addition, we performed Western blot analysis of the key Ca(2+)-handling proteins SERCA2, calsequestrin, phospholamban and the Na(+)/Ca(2+) exchanger. Our major findings were the following: (1) In contrast to the mature myocardium, hESC-CM exhibit negative force-frequency relationships and do not present postrest potentiation. (2) Ryanodine and thapsigargin do not affect the [Ca(2+)](i) transient and contraction, suggesting that, at this developmental stage, the contraction does not depend on sarcoplasmic reticulum Ca(2+) release. (3) In agreement with the finding that a voltage-dependent Ca(2+) current is present in hESC-CM and contributes to the mechanical function, verapamil completely blocks contraction. (4) Although hESC-CM express SERCA2 and Na(+)/Ca(2+) exchanger at levels comparable to those of the adult human myocardium, calsequestrin and phospholamban are not expressed. (4) In agreement with other reports, hESC-CM are responsive to beta-adrenergic stimulation. These findings show that the mechanical function related to intracellular Ca(2+) handling of hESC-CM differs from the adult myocardium, probably because of immature sarcoplasmic reticulum capacity.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.