Anne-Marie Strömberg

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.

Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor

BACKGROUND Human embryonic stem cells (hESCs) have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since current methods, which include slow-freezing-rapid thawing and vitrification of colonies in suspension, present poor viability and high differentiation rates. Dissociated hESC suspensions do not survive cryopreservation because they are susceptible to apoptosis upon cell detachment and dissociation. A selective Rho-associated kinase (ROCK) inhibitor has been reported to increase the survival of dissociated hESCs and their cloning efficiency. METHODS AND RESULTS Here, we describe a novel method for dissociated hESCs cryopreservation in the presence of the ROCK inhibitor Y-27632. The addition of this inhibitor to the freezing and post-thawing medium significantly increased the survival rate and efficiency of colony formation. Moreover, the hESC colonies obtained after the cryopreservation in the presence of the ROCK inhibitor showed a very low rate of differentiation and a reduced time of recovery. After prolonged culture of frozen-thawed dissociated hESCs, the characteristic properties of pluripotent cells were observed, including normal karyotype, morphological features, marker expression (SSEA-4, TRA-1-60, TRA-1-81 and Oct-4) and the potential to differentiate into derivatives of all three germ layers after embryoid bodies formation. CONCLUSION This novel method for the cryopreservation of dissociated hESCs may reduce the time required to amplify frozen stocks, and facilitate not only the storage of large numbers of hESCs but also the widespread use of these cells in regenerative medicine.

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.

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.

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.

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 iPSCs.

Costimulation Blockade Induces Tolerance to HESC Transplanted to the Testis and Induces Regulatory T‐Cells to HESC Transplanted into the Heart

In order to study the ability of costimulation blockade to induce tolerance to human embryonic stem cells (HESC), severe combined immunodeficient (SCID), and immunocompetent C57BL/6 mice treated with costimulation blockade received intratesticular and intramyocardial HESC transplants. All SCID mice with intratesticular HESC transplants developed teratoma. When SCID mice were transplanted intramyocardially, only two of five mice developed teratoma‐like tumors. C57BL/6 mice transplanted intratesticularly and treated with costimulation blockade all developed teratoma and were surrounded by CD4+CD25+Foxp3+ T‐cells, while isotype control treated recipients rejected their grafts. Most C57BL/6 mice transplanted intramyocardially and treated with costimulation blockade demonstrated lymphocytic infiltrates 1 month after transplantation, whereas one maintained its graft. Isolation of regulatory T‐cells from intramyocardial transplanted recipients treated with costimulation blockade demonstrated specificity toward undifferentiated HESC and down‐regulated naive T‐cell activation toward HESC. These results demonstrate that costimulation blockade is sufficiently robust to induce tolerance to HESC in the immune‐privileged environment of the testis. HESC specific regulatory T‐cells developed to HESC transplanted to the heart and the success of transplantation was similar to that seen in SCID mice.

Derivation of 30 human embryonic stem cell lines—improving the quality

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process, we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal, but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked, and they are available for researchers.

β-Amyloid 1-42 Oligomers Impair Function of Human Embryonic Stem Cell-Derived Forebrain Cholinergic Neurons

Cognitive impairment in Alzheimers disease (AD) patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs). Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ) peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES) cells with nerve growth factor (NGF) as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations) and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1–40 increased the number of functional neurons, whereas oligomeric Aβ1–42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1–40 and Aβ1–42 induced gliogenesis. These findings indicate that Aβ1–42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.