Challenges for the Therapeutic use of Pluripotent Stem Derived Cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are an attractive cell source for regenerative medicine. These cells can be expanded to vast numbers and can be differentiated to many desired pluripotent stem cells (PSC) derived therapeutic cells. Cell replacement bears promises, but also challenges. The introduction of exogenous cells in a recipient must address several different topics; its safety, the exclusion of tumor formation, the immunological response and possible rejection, the cells cleanliness and their biological quality, and quantity representing the functionality of the PSC derived therapeutic cells. Tumor formation requires the removal of any PSC remaining after differentiation. Immunological rejection can be addressed with immunomodulation of the cells and the recipient. Cleanliness can be optimized using good manufacturing practice quality systems. At last, the functionality of the cells must be tested in in vitro and in animal models. After addressing these challenges, precise strategies are developed to monitor the status of the cells at different times and in case of undesired results, corresponding counteracting strategies must exist before any clinical attempt

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

The first derivation of human embryonic stem cells (hESCs) (Thomson et al., 1998) and the more recently development of human induced pluripotent stem cells (iPSCs) (Park et al., 2008; Takahashi et al., 2007; Takahashi & Yamanaka, 2006; Wernig et al., 2007; Yu et al., 2007) have marked the beginning of a new era in biomedical research. These two types of human pluripotent stem cells (hPSCs) are characterized by an unlimited capacity to selfrenew while retaining their potential to differentiate into almost all cell types of the body (Odorico et al., 2001; Reubinoff et al., 2000; Silva & Smith, 2008). These remarkable properties turn hPSCs into one of the most interesting cell types for toxicology and drug discovery, tissue engineering and regenerative medicine (Battey, 2007; Mountford, 2008). In fact, work with hPSCs has already provided new and exciting developments that may eventually lead to the creation of novel cell-based therapies for the treatment of a wide range of human diseases including Parkinson’s and other neurodegenerative diseases, diabetes, cardiac and vascular diseases (Kiskinis & Eggan, 2010; Ronaghi et al., 2010). However, a major challenge for the widespread application of hPSCs is the development of efficient protocols for cryopreservation..

The zebrafish transcriptome during early development

<p>Abstract</p> <p>Background</p> <p>The transition from fertilized egg to embryo is accompanied by a multitude of changes in gene expression, and the transcriptional events that underlie these processes have not yet been fully characterized. In this study RNA-Seq is used to compare the transcription profiles of four early developmental stages in zebrafish (<it>Danio rerio</it>) on a global scale.</p> <p>Results</p> <p>An average of 79 M total reads were detected from the different stages. Out of the total number of reads 65% - 73% reads were successfully mapped and 36% - 44% out of those were uniquely mapped. The total number of detected unique gene transcripts was 11187, of which 10096 were present at 1-cell stage. The largest number of common transcripts was observed between 1-cell stage and 16-cell stage. An enrichment of gene transcripts with molecular functions of DNA binding, protein folding and processing as well as metal ion binding was observed with progression of development. The sequence data (accession number ERP000635) is available at the European Nucleotide Archive.</p> <p>Conclusion</p> <p>Clustering of expression profiles shows that a majority of the detected gene transcripts are present at steady levels, and thus a minority of the gene transcripts clusters as increasing or decreasing in expression over the four investigated developmental stages. The three earliest developmental stages were similar when comparing highly expressed genes, whereas the 50% epiboly stage differed from the other three stages in the identity of highly expressed genes, number of uniquely expressed genes and enrichment of GO molecular functions. Taken together, these observations indicate a major transition in gene regulation and transcriptional activity taking place between the 512-cell and 50% epiboly stages, in accordance with previous studies.</p

A gene expression signature shared by human mature oocytes and embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation.</p> <p>Results</p> <p>Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as <it>LIN28 </it>and <it>TDGF1</it>, a large chromatin remodelling network (<it>TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5</it>), 18 different zinc finger transcription factors, including <it>ZNF84</it>, and several still poorly annotated genes such as <it>KLHL7</it>, <it>MRS2</it>, or the Selenophosphate synthetase 1 (<it>SEPHS1</it>). Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome.</p> <p>Conclusion</p> <p>These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.</p

Brain gene expression profiles of Cln1 and Cln5 deficient mice unravels common molecular pathways underlying neuronal degeneration in NCL diseases

<p>Abstract</p> <p>Background</p> <p>The neuronal ceroid lipofuscinoses (NCL) are a group of children's inherited neurodegenerative disorders, characterized by blindness, early dementia and pronounced cortical atrophy. The similar pathological and clinical profiles of the different forms of NCL suggest that common disease mechanisms may be involved. To explore the NCL-associated disease pathology and molecular pathways, we have previously produced targeted knock-out mice for <it>Cln1 </it>and <it>Cln5</it>. Both mouse-models replicate the NCL phenotype and neuropathology; the <it>Cln1-/- </it>model presents with early onset, severe neurodegenerative disease, whereas the <it>Cln5-/- </it>model produces a milder disease with a later onset.</p> <p>Results</p> <p>Here we have performed quantitative gene expression profiling of the cortex from 1 and 4 month old <it>Cln1-/- </it>and <it>Cln5-/- mice</it>. Combined microarray datasets from both mouse models exposed a common affected pathway: genes regulating neuronal growth cone stabilization display similar aberrations in both models. We analyzed locus specific gene expression and showed regional clustering of <it>Cln1 </it>and three major genes of this pathway, further supporting a close functional relationship between the corresponding gene products; adenylate cyclase-associated protein 1 (Cap1), protein tyrosine phosphatase receptor type F (Ptprf) and protein tyrosine phosphatase 4a2 (Ptp4a2). The evidence from the gene expression data, indicating changes in the growth cone assembly, was substantiated by the immunofluorescence staining patterns of <it>Cln1-/- </it>and <it>Cln5-/- </it>cortical neurons. These primary neurons displayed abnormalities in cytoskeleton-associated proteins actin and β-tubulin as well as abnormal intracellular distribution of growth cone associated proteins GAP-43, synapsin and Rab3.</p> <p>Conclusion</p> <p>Our data provide the first evidence for a common molecular pathogenesis behind neuronal degeneration in INCL and vLINCL. Since <it>CLN1 </it>and <it>CLN5 </it>code for proteins with distinct functional roles these data may have implications for other forms of NCLs as well.</p

Maternal stress or sleep during pregnancy are not reflected on telomere length of newborns

publishedVersionPeer reviewe

An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells

BACKGROUND Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) bear a great potential in regenerative medicine. In addition to optimized clinical grade culture conditions, efficient clinical grade cryopreservation methods for these cells are needed. Obtaining good survival after thawing has been problematic. METHODS We used a novel, chemically defined effective xeno-free cryopreservation system for cryostorage and banking of hESCs and iPSCs. The earlier established slow freezing protocols have, even after recent improvements, resulted in low viability and thawed cells had a high tendency to differentiate. The medium is a completely serum and animal substance free product containing dimethylsulfoxide, anhydrous dextrose and a polymer as cryoprotectants. The cells were directly frozen at −70°C, without a programmed freezer. RESULTS The number of frozen colonies versus the number of surviving colonies differed significantly for both HS293 (χ2 = 9.616 with one degree of freedom and two-tailed P = 0.0019) and HS306 (χ2 = 8.801 with one degree of freedom and two-tailed P = 0.0030). After thawing, the cells had a high viability (90-96%) without any impact on proliferation and differentiation, compared with the standard freezing procedure where viability was much lower (49%). The frozen-thawed hESCs and iPSCs had normal karyotype and maintained properties of pluripotent cells with corresponding morphological characteristics, and expressed pluripotency markers after 10 passages in culture. They formed teratomas containing tissue components of the three germ layers. CONCLUSION The defined freezing-thawing system described here offers an excellent simple option for banking of hESCs and iPSC

Monitoring and analysis of dynamic growth of human embryonic stem cells : comparison of automated instrumentation and conventional culturing methods

Abstract Background Human embryonic stem cells (hESCs) are a potential source of cells for use in regenerative medicine. Automation of culturing, monitoring and analysis is crucial for fast and reliable optimization of hESC culturing methods. Continuous monitoring of living cell cultures can reveal more information and is faster than using laborious traditional methods such as microscopic evaluation, immunohistochemistry and flow cytometry. Methods We analyzed the growth dynamics of two hESC lines HS237 and HS293 in a conventional culture medium containing serum replacement and a xeno-free X-vivo 10 medium. We used a new automated culture platform utilizing machine vision technology, which enables automatic observation, recording and analysis of intact living cells. We validated the results using flow cytometry for cell counting and characterization. Results In our analyses, hESC colony growth could be continuously monitored and the proportion of undifferentiated cells automatically analyzed. No labeling was needed and we could, for the first time, perform detailed follow up of live, undisturbed cell colonies, and record all the events in the culture. The growth rate of the hESCs cultured in X-vivo 10 medium was significantly lower and a larger proportion of the cells were differentiated. Conclusion The new automated system enables rapid and reliable analysis of undifferentiated growth dynamics of hESCs. We demonstrate the effectiveness of the system by comparing hESC growth in different culture conditions

A teratocarcinoma-like human embryonic stem cell (hESC) line and four hESC lines reveal potentially oncogenic genomic changes

The first Swiss human embryonic stem cell (hESC) line, CH-ES1, has shown features of a malignant cell line. It originated from the only single blastomere that survived cryopreservation of an embryo, and it more closely resembles teratocarcinoma lines than other hESC lines with respect to its abnormal karyotype and its formation of invasive tumors when injected into SCID mice. The aim of this study was to characterize the molecular basis of the oncogenicity of CH-ES1 cells, we looked for abnormal chromosomal copy number (by array Comparative Genomic Hybridization, aCGH) and single nucleotide polymorphisms (SNPs). To see how unique these changes were, we compared these results to data collected from the 2102Ep teratocarcinoma line and four hESC lines (H1, HS293, HS401 and SIVF-02) which displayed normal G-banding result. We identified genomic gains and losses in CH-ES1, including gains in areas containing several oncogenes. These features are similar to those observed in teratocarcinomas, and this explains the high malignancy. The CH-ES1 line was trisomic for chromosomes 1, 9, 12, 17, 19, 20 and X. Also the karyotypically (based on G-banding) normal hESC lines were also found to have several genomic changes that involved genes with known roles in cancer. The largest changes were found in the H1 line at passage number 56, when large 5 Mb duplications in chromosomes 1q32.2 and 22q12.2 were detected, but the losses and gains were seen already at passage 22. These changes found in the other lines highlight the importance of assessing the acquisition of genetic changes by hESCs before their use in regenerative medicine applications. They also point to the possibility that the acquisition of genetic changes by ESCs in culture may be used to explore certain aspects of the mechanisms regulating oncogenesis