Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer.

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon- and transposase-encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.

Cytokine requirements differ for stroma and embryoid body-mediated hematopoiesis from human embryonic stem cells.

OBJECTIVE: Human embryonic stem (ES) cells can be induced to differentiate into hematopoietic lineages either by stromal cell coculture or by formation of embryoid bodies (EBs). Here, we better characterize cell-bound and secreted factors that support this hematopoietic development. METHODS: Human ES cells either cocultured on the mouse bone marrow cell line S17, or allowed to form EBs, were induced to differentiate in the presence of serum, serum-free conditions, and serum-free media supplemented with defined cytokines. To better characterize the requirement for stromal cell-bound or secreted proteins, S17 conditioned media and transwell cultures were also utilized. RESULTS: In both models, CD34(+), CD45(+), and hematopoietic colony-forming cells (CFCs) were routinely derived. While hematopoietic development was diminished without serum, here we demonstrate with the stromal cell coculture model that addition of the growth factors stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 ligand (Flt3L) to serum-free media does allow isolation of hematopoietic progenitors. However, these same three growth factors added to serum-free media do not support significant hematopoiesis in the EB system. However, addition of the mesoderm-inducing factors bone morphogenic protein-4 and vascular endothelial growth factor to EBs grown in serum-free media plus SCF, TPO, and Flt-3L does improve hematopoietic development. CONCLUSION: These results demonstrate the utility of human ES cell to evaluate specific stimuli that regulate cell fate decisions and the survival of specific lineages. Moreover, the method used to promote differentiation of ES cells may alter the cytokines or growth factors required to isolate specific cell types.

Family or friends: who plays a greater supportive role for Latino college students?

This study compared the relative contribution of perceived family and friend support to psychological well-being and distress and examined whether family or friend support moderated the effects of stress on psychological adjustment in 338 Latino (228 Mexican American, 110 Central American) college students from a predominantly Latino university. Two multiple regressions, controlling for gender, socioeconomic level, acculturation level, and stresses (generic college, acculturative, and minority status), showed that friend support made a slightly greater contribution to well-being than family support, and friend support and not family support protected against psychological distress. Neither family nor friend support moderated the effects of stress on psychological adjustment. Further examination of these variables that assess common-specific stresses within a culture-specific theoretical framework is recommended.

Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells.

Human embryonic stem cells (hESCs) provide an important means to effectively study soluble and cell-bound mediators that regulate development of early blood and endothelial cells in a human model system. Here, several complementary methods are used to demonstrate canonical Wnt signaling is important for development of hESC-derived cells with both hematopoietic and endothelial potential. Analyses using both standard flow cy-tometry, as well the more detailed high-throughput image scanning flow cytometry, characterizes sequential development of distinct early developing CD34(bright)CD31(+)Flk1(+) cells and a later population of CD34(dim)CD45(+) cells. While the CD34(bright)CD31(+)Flk1(+) have a more complex morphology and can develop into both endothelial cells and hematopoietic cells, the CD34(dim)CD45(+) cells have a simpler morphology and give rise to only hematopoietic cells. Treatment with dickkopf1 to inhibit Wnt signaling results in a dramatic decrease in development of cells with hematoendothelial potential. In addition, activation of the canonical Wnt signaling pathway in hESCs by coculture with stromal cells that express Wnt1, but not use of noncanonical Wnt5-expressing stromal cells, results in an accelerated differentiation and higher percentage of CD34(bright)CD31(+)Flk1(+) cells at earlier stages of differentiation. These studies effectively demonstrate the importance of canonical Wnt signaling to mediate development of early hematoendothelial progenitors during human development.

Lessons from an evaluation of a boater outreach program for manatee protection.

Watercraft collisions account for 25-30% of manatee deaths annually in Florida. Education and outreach interventions for boaters are strategies for reducing manatee mortality. We evaluated the effectiveness of the Manatee Watch program by surveying primary boat users whose boats were approached by Manatee Watch. We compared the attitudes, knowledge, and behavioral intentions of boaters who received Manatee Watch materials with a control group of boaters observed by the Florida Marine Research Institute in Tampa Bay during 1999-2001. Results of the 51-item telephone survey with 499 boaters indicated that the Manatee Watch intervention had little effect on boater's attitudes, knowledge, and behaviors regarding manatees. However, individual attitude scores were positively correlated with safe boating behaviors in shallow waters including maintaining a slower speed and watching out for manatees. Overall knowledge about manatees was correlated with one manatee-safe boating behavior. To improve efficacy, educators should (a) incorporate evaluation into the planning stages of program development; (b) target messages to influence boaters' attitudes toward manatees and ecosystem health, and their feelings of ownership and empowerment; (c) facilitate active participation of the boaters; and (d) increase the duration and variety of intervention.

Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity.

Human embryonic stem cells (hESCs) provide an important means to characterize early stages of hematopoietic development. However, the in vivo potential of hESC-derived hematopoietic cells has not been well defined. We demonstrate that hESC-derived cells are capable of long-term hematopoietic engraftment when transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human CD45(+) and CD34(+) cells are identified in the mouse bone marrow (BM) more than 3 months after injection of hESCs that were allowed to differentiate on S17 stromal cells for 7-24 days. Secondary engraftment studies further confirm long-term repopulating cells derived from hESCs. We also evaluated two mechanisms that may inhibit engraftment: host immunity and requirement for homing to BM. Treatment with anti-ASGM1 antiserum that primarily acts by depletion of natural killer cells in transplanted mice leads to improved engraftment, likely due to low levels of HLA class I expressed on hESCs and CD34(+) cells derived from hESCs. Intra-BM injection also provided stable engraftment, with hematopoietic cells identified in both the injected and contra-lateral femur. Importantly, no teratomas are evident in animals injected with differentiated hESCs. These results demonstrate that SCID-repopulating cells, a close surrogate for hematopoietic stem cells, can be derived from hESCs. Moreover, both adaptive and innate immune effector cells may be barriers to engraftment of these cells.

Understanding barriers to implementation of an adaptive land management program.

The Florida Fish and Wildlife Conservation Commission manages over 650,000 ha, including 26 wildlife management and environmental areas. To improve management, they developed an objective-based vegetation management (OBVM) process that focuses on desired conditions of plant communities through an adaptive management framework. Our goals were to understand potential barriers to implementing OBVM and to recommend strategies to overcome barriers. A literature review identified 47 potential barriers in six categories to implementation of adaptive and ecosystem management: logistical, communication, attitudinal, institutional, conceptual, and educational. We explored these barriers through a bureau-wide survey of 90 staff involved in OBVM and personal interviews with area managers, scientists, and administrators. The survey incorporated an organizational culture assessment instrument to gauge how institutional factors might influence OBVM implementation. The survey response rate was 69%. Logistics and communications were the greatest barriers to implementing OBVM. Respondents perceived that the agency had inadequate resources for implementing OBVM and provided inadequate information. About one-third of the respondents believed OBVM would decrease their job flexibility and perceived greater institutional barriers to the approach. The 43% of respondents who believed they would have more responsibility under OBVM also had greater attitudinal barriers. A similar percentage of respondents reported OBVM would not give enough priority to wildlife. Staff believed that current agency culture was hierarchical but preferred a culture that would provide more flexibility for adaptive management and would foster learning from land management activities. In light of the barriers to OBVM, we recommend the following: (1) mitigation of logistical barriers by addressing real and perceived constraints of staff, funds, and other resources in a participatory manner; (2) mitigation of communication barriers through interpersonal and electronic communication channels; (3) development of an OBVM external advisory committee; and (4) adoption of characteristics of an organizational culture that promotes flexibility and learning.