Challenges for the Therapeutic use of Pluripotent Stem Derived Cells

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) are an attractive cell source for regenerative medicine. These cells can be expanded to vast numbers and can be differentiated to many desired pluripotent stem cells (PSC) derived therapeutic cells. Cell replacement bears promises, but also challenges. The introduction of exogenous cells in a recipient must address several different topics; its safety, the exclusion of tumor formation, the immunological response and possible rejection, the cells cleanliness and their biological quality, and quantity representing the functionality of the PSC derived therapeutic cells. Tumor formation requires the removal of any PSC remaining after differentiation. Immunological rejection can be addressed with immunomodulation of the cells and the recipient. Cleanliness can be optimized using good manufacturing practice quality systems. At last, the functionality of the cells must be tested in in vitro and in animal models. After addressing these challenges, precise strategies are developed to monitor the status of the cells at different times and in case of undesired results, corresponding counteracting strategies must exist before any clinical attempt

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 iPSC

Improving Drama Script Writing through Modeling Strategies

  This action research refers to Elliot model (1991)  that aims to improve the ability of writing a screenplay through modeling strategies. This action researchon SMA of Purbolinggo Lampung in the academic year of  2014/2015. The implementation of this research is three cycles. Each implementation of actions is divided into stages, identifying intial idea, reconnaissance, general planning, implement of action, monitor implemention,   reflection, reconnaissance and revise general idea. The results of the reflection activities function as  a foothold to draw up a plan of action to the next cycle. The research results can be concluded: the first process of improving writing skills through drama script modeling strategy. Second, the results of the upgrade script writing drama through modeling strategies, ranging from initial tests as exploitation of the capabilities of the beginning to the end of the test third cycle  the experience increased. (1) The results of initial tests average score 40.1 ((level less); (2) the first cycle  the average score 42.4 (level less); (3) the second cycle  the average score 64.3 (level of being); and (4) the third cycle the average score 83.4. This score has already reached a high level

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

An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells

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

Distinct differentiation characteristics of individual human embryonic stem cell lines

BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. CONCLUSION: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state

Changing Patient and Public Beliefs About Antimicrobials and Antimicrobial Resistance (AMR) Using a Brief Digital Intervention.

BBackground: A key driver of antimicrobial resistance (AMR) is patient demand for unnecessary antibiotics, which is driven by patients’ beliefs about antibiotics and AMR. Few interventions have targeted beliefs to reduce inappropriate demand. Objective: To examine whether a brief, online algorithm-based intervention can change beliefs that may lead to inappropriate antibiotic demand (i.e. perceptions of antibiotic necessity and lack of concern about antibiotic harm). Design: Pre- and post-intervention study. Participants: Participants were 18 years or older, and residing in the United Kingdom, who self-selected to participate via Amazon mTurk, an online survey plaform, and via research networks. Intervention: Participants were presented with a hypothetical situation of cold and flu symptoms, then exposed to the intervention. The online intervention comprised: 1) a profiling tool identifying individual beliefs (antibiotic necessity, concerns, and knowledge) driving inappropriate antibiotic demand; 2) messages designed to change beliefs and knowledge (i.e. reduce antibiotic necessity, and increase antibiotic concerns and knowledge), and 3) an algorithm linking specific messages to specific beliefs and knowledge. Main measures: The profiling tool was repeated immediately after the intervention and compared with baseline scores to assess change in beliefs. A paired samples t-test was used to determine intervention effect. Key Results: A total of 100 respondents completed the study. A significant change in beliefs relating to inappropriate demand was observed after the intervention, with a reduction in beliefs about antibiotic necessity (t = 7.254; p < 0.0001), an increase in antibiotic concerns (t = −7.214; p < 0.0001), and increases in antibiotic and AMR knowledge (t = −4.651; p < 0.0001). Conclusion: This study is the first to demonstrate that patient beliefs about antibiotics and AMR associated with inappropriate demand can be changed by a brief, tailored online intervention. This has implications for the design of future interventions to reduce unnecessary antimicrobial use

Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells

BACKGROUND Feeder cells are frequently used for the early-stage of derivation and culture of human embryonic stem cell (hESC) lines. METHODS We established a conditionally immortalized human foreskin fibroblast line that secreted basic fibroblast growth factor (bFGF). These cells were used as feeder cells for hESC culture and induced pluripotent stem (iPS) cell derivation and expansion. This conditional immortalization was performed using lentiviral vector (LV) mediated transduction of Bmi-1 and human telomerase reverse transcriptase genes and the resulting cell line was further modified by LV-mediated transduction of a secreted form of bFGF gene product. Three different laboratories have tested whether this feeder cell line could support the maintenance of four different hESC lines. RESULTS Immortalized fibroblasts secreting stable amounts of bFGF supported the growth of all hESC lines, which remained pluripotent and had a normal karyotype for at least 10 passages. Even at high passage (p56), these modified cells, when used as feeders, could support iPS derivation and propagation. Derived iPS cells expressed pluripotency markers, had hESC morphology and produced tissue components of the three germ layers when differentiated in vitro. CONCLUSION These modified fibroblasts are useful as a genetically-defined feeder cell line for reproducible and cost-effective culture of both hESC and iPS cell