José Inzunza

Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511

We describe a system for culturing human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells on a recombinant form of human laminin-511, a component of the natural hES cell niche. The system is devoid of animal products and feeder cells and contains only one undefined component, human albumin. The hES cells self-renewed with normal karyotype for at least 4 months (20 passages), after which the cells could produce teratomas containing cell lineages of all three germ layers. When plated on laminin-511 in small clumps, hES cells spread out in a monolayer, maintaining cellular homogeneity with approximately 97% OCT4-positive cells. Adhesion of hES cells was dependent on α6β1 integrin. The use of homogeneous monolayer hES or iPS cell cultures provides more controllable conditions for the design of differentiation methods. This xeno-free and feeder-free system may be useful for the development of cell lineages for therapeutic purposes.

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.

Estrogen Receptor β Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERα and ERβ. ERα mediates the proliferative effect of estrogen in breast cancer cells, whereas ERβ seems to be antiproliferative. We engineered ERα-positive T47D breast cancer cells to express ERβ in a Tet-Off–regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERβ. The presence of ERβ resulted in a reduction in tumor growth. Comparison of the ERβ-expressing and non-ERβ–expressing tumors revealed that the expression of ERβ caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor β (PDGFβ). In cell culture, with the Tet-Off–regulated ERβ-expressing cells, expression of ERβ decreased expression of VEGF and PDGFβ mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFβ proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFβ promoter constructs revealed a repressive effect of ERβ at the promoter level of these genes. Taken together, these data show that introduction of ERβ into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis. (Cancer Res 2006; 66(23): 11207-13)

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.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

A high degree of aneuploidy in frozen-thawed human preimplantation embryos

Abstract We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed.

Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor

Human embryonic stem (hES) cells have traditionally been cultured in medium containing fetal calf serum (FCS) and mouse fibroblasts as feeder cells. The use of animal derived materials carries a risk of transmitting animal pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. This technical study aiming at facilitating IVF units to establish new hES cell lines, has systematically compared the non-differentiated growth of the hES cell line HS237, originally derived and thereafter cultured using human foreskin fibroblasts as feeder cells, by culturing it in media containing serum replacement (SR; 10, 15, 20%), FCS, and human serum. In addition, optimal concentrations of insulin-transferrin-selenium (ITS) mixture and the effect of basic fibroblast growth factor (bFGF) have also been studied. Cellular growth was monitored daily and maintenance of their non-differentiated character was studied using antibodies against TRA-1-60, TRA-1-81 and SSEA-4 and expression of Oct-4. The hES cells proliferated fastest when 20% of SR was used. In human serum-containing medium, the cells underwent extensive spontaneous differentiation within a few passages. The FCS supported the non-differentiated growth poorly. Basic fibroblast growth factor supported non-differentiated growth, the highest concentration (8 ng/ml) giving the best result, while ITS was not beneficial.

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.

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.

Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers

We have developed preimplantation genetic diagnosis (PGD) for carriers of chromosomal abnormalities using fluorescent in situ hybridisation (FISH). Here we present the detailed analysis of 64 biopsied, normally developing embryos obtained from four Robertsonian and three reciprocal translocation carriers in 11 treatment cycles of which four resulted in normal pregnancies (three simplex, one duplex). In order to investigate the degree of mosaicism and segregation mode in the embryos, the primary analysis of the biopsied cells was extended with the analysis of all cells from the non‐transferred embryos. The analysis also included a second hybridisation with two additional probes, not involved in the translocation (chromosomes 1 and 9), in order to investigate the overall degree of mosaicism. Seventeen out of 64 analysed embryos were balanced for the chromosomes involved in the translocation and 14 of these were transferred. Forty‐seven out of 64 embryos (73%) were mosaic regarding the chromosomes involved in the translocation and alternate segregation mode was the most common mode of segregation. Moreover, we have found a higher degree of mosaicism for the chromosomes involved in translocations as compared to control chromosomes. This difference was more pronounced for the embryos from reciprocal translocation carriers. The results, mechanisms, significance and implications of our findings are discussed. Copyright