Heli Skottman

Derivation of Human Embryonic Stem Cell Lines in Serum Replacement Medium Using Postnatal Human Fibroblasts as Feeder Cells

Derivation and culture of human embryonic stem cells (hESCs) without animal‐derived material would be optimal for cell transplantation. We derived two new hES (HS293 and HS306) and 10 early cell lines using serum replacement (SR) medium instead of conventional fetal calf serum and human foreskin fibroblasts as feeder cells. Line HS293 has been in continuous culture, with a passage time of 5–8 days, since October 2003 and is at passage level 56. Line HS306 has been cultured since February 2004, now at passage 41. The lines express markers of pluripotent hESCs (Oct‐4, SSEA‐4, TRA‐1‐60, TRA‐1‐81, GCTM‐2, and alkaline phosphatase). The pluripotency has been shown in embryoid bodies in vitro, and the pluripotency of line 293 has also been shown in vivo by teratoma formation in severe combined immunodeficiency/beige mice. The karyotype of HS293 is 46,XY, and that of HS306 is 46,XX. Ten more early lines have been derived under similar conditions since September 2004. We conclude that hESC lines can be successfully derived using SR medium and postnatal human fibroblasts as feeder cells. This is a step toward xeno‐free conditions and facilitates the use of these cells in transplantation.

Good manufacturing practice and clinical-grade human embryonic stem cell lines

Human embryonic stem cell (hESC) lines, after directed differentiation, hold the greatest potential for cell transplantation treatment in many severe diseases. Good manufacturing practice (GMP) quality, defined by both the European Medicines Agency and the Food and Drug Administration, is a requirement for clinical-grade cells, offering optimal defined quality and safety in cell transplantation. Using animal substance-free culture media, feeder cells or feeder-free matrix in derivation, passaging, expansion and cryopreservation procedures, immune reactions against animal proteins in the cells, and infection risk caused by animal microbes can be avoided. It is also possible to apply GMP to animal components if no better options are available. In recent production of GMP-quality hESC lines, feeder cells had been cultured in fetal bovine serum, and the medium supplemented with an animal protein containing a serum replacement component. Using embryos cultured in a GMP laboratory, isolating the inner cell mass mechanically, deriving lines on human feeder cells originally cultured in xeno-free medium in a GMP laboratory, and using xeno-free media for derivation and culture of hESC lines themselves, GMP-quality xeno-free hESC lines could be established today. Human serum is a xeno-free component available today, but many chemically defined media are under development.

Gene expression signatures of seven individual human embryonic stem cell lines.

Identification of molecular components that define a pluripotent human embryonic stem cell (hESC) provides the basis for understanding the molecular mechanisms regulating the maintenance of pluripotency and induction of differentiation. We compared the gene expression profiles of seven genetically independent hESC lines with those of nonlineage‐differentiated cells derived from each line. A total of 8,464 transcripts were expressed in all hESC lines. More than 45% of them have no yet‐known biological function, which indicates that a high number of unknown factors contribute to hESC pluripotency. Among these 8,464 transcripts, 280 genes were specific for hESCs and 219 genes were more than twofold differentially expressed in all hESC lines compared with nonlineage‐differentiated cells. They represent genes implicated in the maintenance of pluripotency and those involved in early differentiation. The chromosomal distribution of these hESC‐enriched genes showed over‐representation in chromosome 19 and under‐representation in chromosome 18. Although the overall gene expression profiles of the seven hESC lines were markedly similar, each line also had a subset of differentially expressed genes reflecting their genetic variation and possibly preferential differentiation potential. Limited overlap between gene expression profiles illustrates the importance of cross‐validation of results between different ESC lines.

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells

Background The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable. Methodology/Principal Findings Here, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed. Conclusion/Significance Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.

Human embryonic stem cells are immunogenic in allogeneic and xenogeneic settings

Recent studies have suggested that human embryonic stem cells (HESC) are immune-privileged and may thereby circumvent rejection. The expression of immunologically active molecules was studied by DNA microarray analysis and by flow cytometry. HESC were transplanted into immunologically competent mice and traced by fluorescence in-situ hybridization (FISH) and immunohistochemistry. The ability of HESC to directly and indirectly induce immune responses in CD4+ T-cells from naive and transplanted mice was studied. Their ability to induce immune responses of human CD4+ T-cells, when cultured in the presence of dendritic cells (DC) syngeneic to responder T-cells, was also analysed. HESC demonstrated expression of HLA class I and HLA class II genes, but the cell surface expression of HLA class II molecules was low even after incubation with IFNgamma. In wild-type mice, HESC could be demonstrated by FISH until 3 days after transplantation and were surrounded by heavy infiltrates of T-cells and macrophages. HESC induced a similar immune response as human fibroblast cells (HFib) on naive and immunized T-cells, both directly and in the presence of syngeneic DC. A similar response was observed in the allogeneic setting. It is concluded that HESC are immunologically inert and do not inhibit immune responses during direct or indirect antigen presentation, and they were acutely rejected in a xenogeneic setting.

Transcriptome Profiling of Human Pre-Implantation Development

Background Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known. Methodology By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development. Principal Findings We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution. Significance Our database and findings provide fundamental resources for understanding the genetic network controlling early human development.

CD marker expression profiles of human embryonic stem cells and their neural derivatives, determined using flow-cytometric analysis, reveal a novel CD marker for exclusion of pluripotent stem cells.

Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation.

Comparison of Biomaterials and Extracellular Matrices as a Culture Platform for Multiple, Independently Derived Human Embryonic Stem Cell Lines

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins, human and animal sera matrices, and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media, human foreskin fibroblast-conditioned culture medium, chemically defined medium, TeSR1, and modified TeSR1 media. The results showed the undefined, xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins, serum matrices, and the biomaterials tested. A long-term, feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium, but a xeno-free, fully defined, and reproducible feeder cell-free hESC culture method still remains to be developed.

Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro

The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

Unique Gene Expression Signature by Human Embryonic Stem Cells Cultured Under Serum‐Free Conditions Correlates with Their Enhanced and Prolonged Growth in an Undifferentiated Stage

Understanding the interaction between human embryonic stem cells (hESCs) and their microenvironment is crucial for the propagation and the differentiation of hESCs for therapeutic applications. hESCs maintain their characteristics both in serum‐containing and serum‐replacement (SR) media. In this study, the effects of the serum‐containing and SR culture media on the gene expression profiles of hESCs were examined. Although the expression of many known embryonic stem cell markers was similar in cells cultured in either media, surprisingly, 1,417 genes were found to be differentially expressed when hESCs cultured in serum‐containing medium were compared with those cultured in SR medium. Several genes upregulated in cells cultured in SR medium suggested increased metabolism and proliferation rates in this medium, providing a possible explanation for the increased growth rate of nondifferentiated cells observed in SR culture conditions compared with that in serum medium. Several genes characteristic for cells with differentiated phenotype were expressed in cells cultured in serum‐containing medium. Our data clearly indicate that the manipulation of hESC culture conditions causes phenotypic changes of the cells that were reflected also at the level of gene expression. Such changes may have fundamental importance for hESCs, and gene expression changes should be monitored as a part of cell culture optimization aiming at a clinical use of hESCs for cell transplantation.