Supporting a vibrant entrepreneurial ecosystem: High density, critical mass, and effective strategies underpin the success of innovative life science ecosystems.

How do you create a vibrant entrepreneurial life science ecosystem? How research communities in the Rhine-Main-Neckar region around Heidelberg have grown and flourished.

Derivation of Huntington Disease affected Genea091 human embryonic stem cell line.

The Genea091 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 40 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, 92% of cells expressed Nanog, 97% Oct4, 79% Tra1-60 and 98% SSEA4 and gave a Pluritest pluripotency score of 38.36, Novelty of 1.35. The cell line was negative for Mycoplasma and visible contamination.

Derivation of DM2 affected human embryonic stem cell line Genea066.

The Genea066 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying expansion of CCTG repeats in exon 1 of the ZNF9 gene, indicative of Myotonic Dystrophy Type 2 (DM2). 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, 88% of cells expressed Nanog, 97% Oct4, 80% Tra1-60 and 99% SSEA4 and gave a Pluritest Pluripotency score of 31.3, Novelty of 1.22. The cell line was negative for Mycoplasma and visible contamination.

Derivation of Huntington Disease affected Genea090 human embryonic stem cell line.

The Genea090 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying Htt gene CAG expansion of 45 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, 91% of cells expressed Nanog, 95% Oct4, 90% Tra1-60 and 100% SSEA4 and gave a pluritest pluripotency score of 30.91, novelty of 1.23. The cell line was negative for Mycoplasma and visible contamination.

Derivation of FSHD1 affected human embryonic stem cell line Genea096.

The Genea096 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying a deletion in 4q35 with only 6 D4Z4 repeats by PGD linkage analysis, indicative of FSHD1. 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, 64% of cells expressed Nanog, 93% Oct4, 58% Tra1-60 and 93% SSEA4 and a Pluritest Pluripotency score of 39.41, Novelty of 1.25. The cell line was negative for Mycoplasma and visible contamination.

Derivation of Human Embryonic Stem Cell Lines from Vitrified Human Blastocysts.

Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have become excess to clinical needs in assisted reproduction programs. They provide cellular models for embryonic development and disease, and are thought to be useful for future cell replacement therapies and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines. This includes blastocyst cryopreservation using a highly efficient vitrification protocol, the production and use of fibroblast feeder cells, embryo plating and passaging of resulting cellular outgrowths, and cryopreservation of putative stem cells lines.

Generation of human embryonic stem cells.

This unit describes generation of human embryonic stem cell lines from early human embryos. The focus is on actual handling of embryos and early embryonic outgrowths, omitting steps required for actual generation, freezing, and thawing of embryos, as well as further culture and characterization of newly derived stem cells. Hence, the initial culture of embryos to a blastocyst stage is described, followed by removal of the protective zona pellucida layer, isolation of the inner cell mass (ICM), subsequent plating of ICM or whole embryo and, finally, the first few passages of an early embryonic outgrowth. A few alternative procedures for some steps such as zona removal and inner cell mass isolation are described, to allow procedures to be modified according to circumstances.

Derivation of human embryonic stem cell lines from vitrified human embryos.

Human embryonic stem cell lines are usually derived from human embryos that have become excess to clinical needs in assisted reproduction programs, whether because the couple in question has completed their family or because the embryo was found to be clinically unsuitable for transfer due to severe genetic condition (in case of pre-implantation genetic diagnosis, PGD). Culturing embryos to a blastocyst stage (5-6 days after IVF) before embryo transfer or cryopreservation instead of earlier commonly used 8-cell stage (3 days after IVF) calls for new methods for embryo cryopreservation and allows higher efficiencies for the actual stem cell derivation. Despite the vast advances in other fields of embryonic stem cell research, methods for derivation of new lines have not changed much over the years, mainly due to scarcity of embryos limiting experimentation. We describe here methods required to derive new embryonic stem cell lines starting from the initial cryopreservation of an embryo and finishing with a new cell line. We cover embryo cryopreservation and warming using a highly efficient vitrification method, the production of feeder cells and feeder plates, as well as embryo handling, plating and critical early passages, including earliest possible cryopreservation of putative stem cells using vitrification.

Increased apoptosis and skewed differentiation in mouse embryonic stem cells lacking the histone methyltransferase Mll2.

Epigenetic regulation by histone methyltransferases provides transcriptional memory and inheritable propagation of gene expression patterns. Potentially, the transition from a pluripotent state to lineage commitment also includes epigenetic instructions. The histone 3 lysine 4 methyltransferase Mll2/Wbp7 is essential for embryonic development. Here, we used embryonic stem (ES) cell lines deficient for Mll2 to examine its function more accurately. Mll2-/- ES cells are viable and retain pluripotency, but they display cell proliferation defects due to an enhanced rate of apoptosis. Apoptosis was not relieved by caspase inhibition and correlated with decreased Bcl2 expression. Concordantly, Mll2 binds to the Bcl2 gene and H3K4me(3) levels are reduced at the binding site when Mll2 is absent. In vitro differentiation showed delays along representative pathways for all three germ layers. Although ectodermal delays were severe and mesodermal delays persisted at about three days, endodermal differentiation seemed to recover and overshoot, concomitant with prolonged Oct4 gene expression. Hence, Mll2 is not required for ES cell self-renewal or the complex changes in gene expression involved in lineage commitment, but it contributes to the coordination and timing of early differentiation decisions.

Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development.

Epigenesis is the process whereby the daughters of a dividing cell retain a chromatin state determined before cell division. The best-studied cases involve the inheritance of heterochromatic chromosomal domains, and little is known about specific gene regulation by epigenetic mechanisms. Recent evidence shows that epigenesis pivots on methylation of nucleosomes at histone 3 lysines 4, 9 or 27. Bioinformatics indicates that mammals have several enzymes for each of these methylations, including at least six histone 3 lysine 4 methyltransferases. To look for evidence of gene-specific epigenetic regulation in mammalian development, we examined one of these six, Mll2, using a multipurpose allele in the mouse to ascertain the loss-of-function phenotype. Loss of Mll2 slowed growth, increased apoptosis and retarded development, leading to embryonic failure before E11.5. Using chimera experiments, we demonstrated that Mll2 is cell-autonomously required. Evidence for gene-specific regulation was also observed. Although Mox1 and Hoxb1 expression patterns were correctly established, they were not maintained in the absence of Mll2, whereas Wnt1 and Otx2 were. The Mll2 loss-of-function phenotype is different from that of its sister gene Mll, and they regulate different Hox complex genes during ES cell differentiation. Therefore, these two closely related epigenetic factors play different roles in development and maintain distinct gene expression patterns. This suggests that other epigenetic factors also regulate particular patterns and that development entails networks of epigenetic specificities.

A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles.

Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.