Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells.

BACKGROUND AIMS: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. METHODS: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. RESULTS: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. CONCLUSIONS: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

Karolinska Institutet Human Embryonic Stem Cell Bank.

The Karolinska Institutet Human Embryonic Stem Cell Bank (KI Stem Cell Bank) was established at KI, Stockholm, Sweden, when the first human embryonic stem cell (hESC) line was derived by Professor Hovatta and colleagues in 2002. Since then, the bank has grown to include 60 hESC lines. From the very beginning the aim of the bank has been derivation of hESC lines suitable for clinical use. Step by step progress has been made towards this goal, including removal of xeno components, establishment of chemically defined conditions and Good Manufacturing Practice (GMP) compliancy. Today our bank includes such clinical grade hESC line, KARO1, derived and banked according to GMP guidelines. Many of the hESC lines in the bank have been distributed to the scientific community and are deposited in the Stockholm Medical Biobank available for research on collaborative basis.

Derivation of Trisomy 21 affected human embryonic stem cell line Genea021.

The Genea021 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Trisomy 21, indicative of Down Syndrome. Following ICM outgrowth on inactivated human feeders, CGH and STR analyses demonstrated a 47, XY, +21 karyotype and male allele pattern. The hESC line had pluripotent cell morphology, 71% of cells expressed Nanog, 84% Oct4, 23% Tra1-60 and 95% SSEA4, gave a Pluritest Pluripotency score of 21.85, Novelty of 1.42, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

Derivation of Huntington Disease affected Genea018 human embryonic stem cell line.

The Genea018 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 46 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 75% of cells expressed Nanog, 91% Oct4, 73% Tra1-60 and 96% SSEA4, gave a Pluritest pluripotency score of 31.12, Novelty of 1.45, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

Derivation of NEM2 affected human embryonic stem cell line Genea078.

The Genea078 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 76% of cells expressed Nanog, 93% Oct4, 67% Tra1-60 and 97% SSEA4 and gave a Pluritest Pluripotency score of 42.18, Novelty of 1.37. The cell line was negative for Mycoplasma and visible contamination.

Derivation of DM1 affected human embryonic stem cell line Genea067.

The Genea067 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying expansion of CTG repeats in the DMPK gene, indicative of Myotonic Dystrophy Type 1 (DM1). Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 97% Oct4, 73% Tra1-60 and 98% SSEA4 and gave a Pluritest Pluripotency score of 25.75, Novelty of 1.46. The cell line was negative for Mycoplasma and visible contamination.

Derivation of FSHD1 affected human embryonic stem cell line Genea050.

The Genea050 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying a deletion in 4q35 with only 5 D4Z4 repeats by PGD linkage analysis, indicative of FSHD1. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 92% of cells expressed Nanog, 97% Oct4, 79% Tra1-60 and 99% SSEA4, gave a Pluritest Pluripotency score of 25.45, Novelty of 1.45 demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination.

A Human Pluripotent Stem Cell Model of Facioscapulohumeral Muscular Dystrophy-Affected Skeletal Muscles.

UNLABELLED: : Facioscapulohumeral muscular dystrophy (FSHD) represents a major unmet clinical need arising from the progressive weakness and atrophy of skeletal muscles. The dearth of adequate experimental models has severely hampered our understanding of the disease. To date, no treatment is available for FSHD. Human embryonic stem cells (hESCs) potentially represent a renewable source of skeletal muscle cells (SkMCs) and provide an alternative to invasive patient biopsies. We developed a scalable monolayer system to differentiate hESCs into mature SkMCs within 26 days, without cell sorting or genetic manipulation. Here we show that SkMCs derived from FSHD1-affected hESC lines exclusively express the FSHD pathogenic marker double homeobox 4 and exhibit some of the defects reported in FSHD. FSHD1 myotubes are thinner when compared with unaffected and Becker muscular dystrophy myotubes, and differentially regulate genes involved in cell cycle control, oxidative stress response, and cell adhesion. This cellular model will be a powerful tool for studying FSHD and will ultimately assist in the development of effective treatments for muscular dystrophies. SIGNIFICANCE: This work describes an efficient and highly scalable monolayer system to differentiate human pluripotent stem cells (hPSCs) into skeletal muscle cells (SkMCs) and demonstrates disease-specific phenotypes in SkMCs derived from both embryonic and induced hPSCs affected with facioscapulohumeral muscular dystrophy. This study represents the first human stem cell-based cellular model for a muscular dystrophy that is suitable for high-throughput screening and drug development.

Managing the potential and pitfalls during clinical translation of emerging stem cell therapies.

We are moving into a new era of stem cell research where many possibilities for treatment of degenerative, chronic and/or fatal diseases and injuries are becoming primed for clinical trial. These reports have led millions of people worldwide to hope that regenerative medicine is about to revolutionise biomedicine: either through transplantation of cells grown in the laboratory, or by finding ways to stimulate a patient's intrinsic stem cells to repair diseased and damaged organs. While major contributions of stem cells to drug discovery, safety and efficacy testing, as well as modelling 'diseases in a dish' are also expected, it is the in vivo use of stem cells that has captured the general public's attention. However, public misconceptions of stem cell potential and applications can leave patients vulnerable to the influences of profit driven entities selling unproven treatments without solid scientific basis or appropriate clinical testing or follow up. This review provides a brief history of stem cell clinical translation together with an overview of the properties, potential, and current clinical application of various stem cell types. In doing so it presents a clearer picture of the inherent risks and opportunities associated with stem cell research translation, and thus offers a framework to help realise invested expectations more quickly, safely and effectively.

Cell surface antigen profiling using a novel type of antibody array immobilised to plasma ion-implanted polycarbonate.

To identify and sort out subpopulations of cells from more complex and heterogeneous assemblies of cells is important for many biomedical applications, and the development of cost- and labour-efficient techniques to accomplish this is warranted. In this report, we have developed a novel array-based platform to discriminate cellular populations based on differences in cell surface antigen expressions. These cell capture microarrays were produced through covalent immobilisation of CD antibodies to plasma ion immersion implantation-treated polycarbonate (PIII-PC), which offers the advantage of a transparent matrix, allowing direct light microscopy visualisation of captured cells. The functionality of the PIII-PC array was validated using several cell types, resulting in unique surface antigen expression profiles. PIII-PC results were compatible with flow cytometry, nitrocellulose cell capture arrays and immunofluorescent staining, indicating that the technique is robust. We report on the use of this PIII-PC cluster of differentiation (CD) antibody array to gain new insights into neural differentiation of mouse embryonic stem (ES) cells and into the consequences of genetic targeting of the Notch signalling pathway, a key signalling mechanism for most cellular differentiation processes. Specifically, we identify CD98 as a novel marker for neural precursors and polarised expression of CD9 in the apical domain of ES cell-derived neural rosettes. We further identify expression of CD9 in hitherto uncharacterised non-neural cells and enrichment of CD49e- and CD117-positive cells in Notch signalling-deficient ES cell differentiations. In conclusion, this work demonstrates that covalent immobilisation of antibody arrays to the PIII-PC surface provides faithful cell surface antigen data in a cost- and labour-efficient manner. This may be used to facilitate high throughput identification and standardisation of more precise marker profiles during stem cell differentiation and in various genetic and disease contexts.

Notch signaling maintains neural rosette polarity.

Formation of the metazoan body plan requires a complex interplay of morphological changes and patterning, and central to these processes is the establishment of apical/basal cell polarity. In the developing nervous system, apical/basal cell polarity is essential for neural tube closure and maintenance of the neural stem cell population. In this report we explore how a signaling pathway important for nervous system development, Notch signaling, impacts on apical/basal cell polarity in neural differentiation. CSL(-/-) mouse embryos, which are devoid of canonical Notch signaling, demonstrated a neural tube phenotype consistent with cell polarity and convergent extension defects, including deficiencies in the restricted expression of apical polarity markers in the neuroepithelium. CSL(-/-) mouse embryonic stem (ES) cells, cultured at low density, behaved as wild-type in the establishment of neural progenitors and apical specification, though progression through rosette formation, an in vitro correlate of neurulation, required CSL for correct maintenance of rosette structure and regulation of neuronal differentiation. Similarly, acute pharmacological inhibition of Notch signaling led to the breakdown of neural rosettes and accelerated neuronal differentiation. In addition to functional Notch signaling, rosette integrity was found to require actin polymerization and Rho kinase (ROCK) activity. Disruption of rosettes through inhibition of actin polymerization or ROCK activity, however, had no effect on neuronal differentiation, indicating that rosette maintenance is not a prerequisite for normal neuronal differentiation. In conclusion, our data indicate that Notch signaling plays a role not only in differentiation, but also in organization and maintenance of polarity during development of the early nervous system.

A critical role for Sox9 in notch-induced astrogliogenesis and stem cell maintenance.

Notch signaling is a key regulator of cell-fate decisions and is essential for proper neuroectodermal development. There, it favors the formation of ectoderm, promotes maintenance of neural stem cells, inhibits differentiation into neurons, and commits neural progenitors to a glial fate. In this report, we explore downstream effects of Notch important for astroglial differentiation. Transient activation of Notch1 during early stages of neuroectodermal differentiation of embryonic stem cells resulted in an increase of neural stem cells, a reduction in neurons, an induction of astroglial cell differentiation, and an induction of neural crest (NC) development. Transient or continuous activation of Notch1 during neuroectodermal differentiation led to upregulation of Sox9 expression. Knockdown of the Notch1-induced Sox9 expression reversed Notch1-induced astroglial cell differentiation, increase in neural stem cells, and the decrease in neurons, whereas the Notch1 effects on NC development were hardly affected by knockdown of Sox9 expression. These findings reveal a critical role for Notch-mediated upregulation of Sox9 in a select set of neural lineage determination steps controlled by Notch.

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells.

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells.

Interactions between Notch- and hypoxia-induced transcriptomes in embryonic stem cells.

Interaction between key signaling mechanisms is important to generate the diversity in signaling output required for proper control of cellular differentiation and function, although the molecular manifestations of such cross-talk are only partially understood. Notch signaling and the cellular response to hypoxia intersect at different points in the signaling cascades, and in this report we analyze the consequences of this cross-talk at the transcriptome level. Mouse ES cells were subjected to various combinations of hypoxia and/or activated Notch signaling, and the transcriptome changes could be grouped into different categories, reflecting various modes of hypoxia and Notch signaling integration. Two principal categories of novel Notch- and hypoxia-induced genes were identified: (i) a larger set of Notch or hypoxic target genes which were induced by one pathway and not significantly affected by the activity status of the other pathway and (ii) a smaller set of genes co-regulated by Notch and hypoxia. In the latter category, we identified genes that were induced by hypoxia and the expression of which was enhanced by active Notch signaling and another group of genes that were induced by Notch and hypoxia independently. Several of the hypoxia- and Notch-induced genes were found to be upregulated in various forms of cancer. Identification of genes co-regulated by the two pathways may provide a molecular platform to better understand the intersection between the two signaling cascades in normal development and cancer.