Guoliang Meng

ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells

BACKGROUND Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently, however, hES cells must be cryopreserved as clumps when using conventional slow freezing protocols, leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing, as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions. METHODS hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C, cells were cultured in mTeSR medium supplemented with 10 microM of Rho-associated kinase inhibitor Y-27632 for 1 day. RESULTS The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P < 0.01). Furthermore, by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction, we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632, hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point.

The ROCK Inhibitor Y-27632 Enhances the Survival Rate of Human Embryonic Stem Cells Following Cryopreservation

After slow freezing, the survival rate of human embryonic stem (hES) cells is poor and inconsistent. The aim of this study was to increase the freeze-thaw survival rate of hES cells by utilizing the ROCK inhibitor Y-27632. hES cell colonies were first treated with Y-27632, followed by collagenase IV and TrypLE Select dissociation whereupon small clumps were slow frozen using 90% Knockout serum replacement and 10% dimethyl sulfoxide. After thawing at 37 C, the clumps were cultured in medium supplemented with 10 microM Y-27632 for 1 day. Our results show that the use of Y-27632 significantly increases the survival of hES cells after thawing compared with that of the control group. Y-27632-treated freeze-thawed hES cells retain morphology, stable karyotype, expression of cell surface markers, and the potential to differentiate into derivatives of all three germ layers after long-term culture. We have concluded that conventional slow freezing with Y-27632 treatment is efficient and convenient for the cryopreservation of hES cells.

Extracellular Matrix Isolated From Foreskin Fibroblasts Supports Long-Term Xeno-Free Human Embryonic Stem Cell Culture

Human embryonic stem (hES) cells hold great promise for application of human cell and tissue replacement therapy. However, the overwhelming majority of currently available hES cell lines have been directly or indirectly exposed to materials containing animal-derived components during their derivation, propagation, and cryopreservation. Unlike feeder-based cultures, which require the simultaneous growth of feeder and stem cells, resulting in mixed cell populations, stem cells grown on feeder-free systems are easily separated from the surface, presenting a pure population of cells for downstream applications. In this study, we have developed a novel method to expand hES cells in xeno-free, feeder-free conditions using 2 different matrices derived from xeno-free human foreskin fibroblasts (XF-HFFs). Using XF-HFF-derived extracellular matrix, together with 100 ng/mL recombinant bFGF-supplemented HEScGRO Basal Medium, long-term xeno-free expansion of hES cells is possible. Resulting hES cells were subjected to stringent tests and were found to maintain ES cell features, including morphology, pluripotency, stable karyotype, and expression of cell surface markers, for at least 20 passages. Xeno-free culturing practices are essential for the translation of basic hES cell research into the clinic. Therefore, the method presented in this study demonstrates that hES cells can be cultured in complete xeno-free conditions without the loss of pluripotency and furthermore, without the possibility of contamination from exogenous sources.

A Novel Method for Generating Xeno-Free Human Feeder Cells for Human Embryonic Stem Cell Culture

Long-term cultures of human embryonic stem (hES) cells require a feeder layer for maintaining cells in an undifferentiated state and increasing karyotype stability. In routine hES cell culture, mouse embryonic fibroblast (MEF) feeders and animal component-containing media (FBS or serum replacement) are commonly used. However, the use of animal materials increases the risk of transmitting pathogens to hES cells and therefore is not optimal for use in cultures intended for human transplantation. There are other limitations with conventional feeder cells, such as MEFs, which have a short lifespan and can only be propagated five to six passages before senescing. Several groups have investigated maintaining existing hES cell lines and deriving new hES cell lines on human feeder layers. However, almost all of these human source feeder cells employed in previous studies were derived and cultured in animal component conditions. Even though one group previously reported the derivation and culture of human foreskin fibroblasts (HFFs) in human serum-containing medium, this medium is not optimal because HFFs routinely undergo senescence after 10 passages when cultured in human serum. In this study we have developed a completely animal-free method to derive HFFs from primary tissues. We demonstrate that animal-free (AF) HFFs do not enter senescence within 55 passages when cultured in animal-free conditions. This methodology offers alternative and completely animal-free conditions for hES cell culture, thus maintaining hES cell morphology, pluripotency, karyotype stability, and expression of pluripotency markers. Moreover, no difference in hES cell maintenance was observed when they were cultured on AF-HFFs of different passage number or independent derivations.

Synergistic Effect of Medium, Matrix, and Exogenous Factors on the Adhesion and Growth of Human Pluripotent Stem Cells Under Defined, Xeno-Free Conditions

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), share the properties of unlimited self-renewal and the capacity to become any cell type in the body, making them well suited for regenerative medicine and cell therapy. So far, almost all hPSC lines have been directly or indirectly exposed to animal-derived products, which would hinder their use for clinical purposes. One of the biggest challenges in this area is to remove animal components from the derivation, propagation, and cryopreservation of hPSCs. Moreover, the presence of undefined components of animal or human origin in culture system may interfere with the interpretation of the effect of exogenous agents on the growth and differentiation of hPSCs and are prone to significant variability. To explore hPSC expansion in defined, xeno-free conditions, 2 different groups of culture systems were used to culture different hESC and hiPSC lines. Our results suggested that (1) medium, matrix, and exogenous factors have synergistic effects on the adhesion and growth of hPSCs; (2) cooperation of exogenous factors including basic fibroblast growth factor, Rho-associated kinase inhibitor (ROCK), and other growth factors is critical for hPSC adhesion and proliferation; (3) basal media have different effects on hPSC attachment to the culture surface; and (4) a medium or matrix component can work synergistically in one culture system, and not at all in another. In this study, we found that Vitronectin/TeSR2 and PDL/HEScGRO (Y-27632) systems were optimal for maintaining the long-term culture of 3 hESC lines and 2 hiPSC lines under defined, xeno-free conditions.

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

BackgroundWe have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers.ResultsHere, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo.ConclusionOn the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.

Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

Derivation of iPSCs in stirred suspension bioreactors.

Induced pluripotent stem cells (iPSCs) are typically derived in adherent culture. Here we report fast and efficient derivation of mouse iPSCs in stirred suspension bioreactors, with and without the use of c-Myc. Suspension-reprogrammed cells expressed pluripotency markers, showed multilineage differentiation in vitro and in vivo, and contributed to the germline in chimeric mice. Suspension reprogramming has the potential to accelerate and standardize iPSC research.

Cartilage tissue engineering identifies abnormal human induced pluripotent stem cells

Safety is the foremost issue in all human cell therapies, but human induced pluripotent stem cells (iPSCs) currently lack a useful safety indicator. Studies in chimeric mice have demonstrated that certain lines of iPSCs are tumorigenic; however a similar screen has not been developed for human iPSCs. Here, we show that in vitro cartilage tissue engineering is an excellent tool for screening human iPSC lines for tumorigenic potential. Although all human embryonic stem cells (ESCs) and most iPSC lines tested formed cartilage safely, certain human iPSCs displayed a pro-oncogenic state, as indicated by the presence of secretory tumors during cartilage differentiation in vitro. We observed five abnormal iPSC clones amoungst 21 lines derived from five different reprogramming methods using three cellular origins. We conclude that in vitro cartilage tissue engineering is a useful approach to identify abnormal human iPSC lines.

Rapid isolation of undifferentiated human pluripotent stem cells from extremely differentiated colonies.

Conventionally, researchers remove spontaneously differentiated areas in human pluripotent stem cell (hPSC) colonies by using a finely drawn glass pipette or a commercially available syringe needle. However, when extreme differentiation occurs, it is inefficient to purify the remaining undifferentiated cells, as these undifferentiated areas are too small to be isolated completely with the mechanical method. Antibodies can be utilized to purify the rare undifferentiated cells; however, this type of purification cannot be used in xeno-free culture systems. To avoid the loss of valuable hPSCs, we developed a novel method to isolate undifferentiated hPSCs from extremely differentiated colonies that could be easily adapted to xeno-free culture conditions. This protocol involves dissecting away differentiated areas, dissociating the remaining colony into clumps, seeding small clumps into new dishes, and picking undifferentiated colonies for expansion. Using this method, we routinely achieve completely undifferentiated colonies in one passage without the use of antibody-based purification.