This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.To establish system for efficient differentiation of hESC into RBCs with potential to use for blood transfusions.To generate erythroid cells from hESCs, as a first step, we cocultured hES cells with OP9 bone marrow stromal cell line for 6 days to induce their differentiation towards CD34+ cells, which included population of CD34+CD43+ hematopoietic progenitors (10-20%), CD34+CD43-KDR+ endothelial cells (up to 60%), and CD34+CD43-KDR- mesenchymal cells (less than 15%). Subsequently CD34+ cells were isolated using magnetic sorting and cultured in non-adherent conditions in SFEM supplemented with SCF, TPO, EPO, IL-3, IL-6, EX-CYTE, insulin, dexamethasone, and transferrin for five days. From the day 6, cells were expanded in the same media without TPO, IL-3 and IL-6. After 10 days of culture, most of the cells were immature CD71+CD235a+ erythroid progenitors expressing high level of embryonic - and fetal -globin, and low level of adult -globin as determined by PCR. Erythroid progenitors continued proliferate in culture for up to 60 days resulting in more than 4000-fold expansion. Following expansion, -globin expression increased and -globin expression decreased and eventually disappeared by the day 50 of culture. Morphologic evaluation of expanded cells revealed homogenous population of erythroid progenitors at different stages of maturation, including erythroblasts and normoblasts. By flow cytometry, essentially all cells were CD71+CD235a+ and CD34- and CD45-, confirming that they represent pure population of erythroid progenitors free of leukocytes. The described experimental system will be useful for the studies of genes regulating erythroid cell expansion, as well as molecular mechanisms regulating globin gene expression and switches during early hematopoietic development as well for large scale production of red blood cells from hESCs for clinical purposes. This research used WNPRC stem cell resources, start up funding and federally approved hES cell lines. No publications yet.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Primate Research Center Grants (P51)
Project #
2P51RR000167-47A1
Application #
7716462
Study Section
Special Emphasis Panel (ZRR1-CM-8 (01))
Project Start
2008-07-23
Project End
2009-04-30
Budget Start
2008-07-23
Budget End
2009-04-30
Support Year
47
Fiscal Year
2008
Total Cost
$40,957
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Kang, HyunJun; Mesquitta, Walatta-Tseyon; Jung, Ho Sun et al. (2018) GATA2 Is Dispensable for Specification of Hemogenic Endothelium but Promotes Endothelial-to-Hematopoietic Transition. Stem Cell Reports 11:197-211
Rhoads, Timothy W; Burhans, Maggie S; Chen, Vincent B et al. (2018) Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys. Cell Metab 27:677-688.e5
Ellis-Connell, Amy L; Balgeman, Alexis J; Zarbock, Katie R et al. (2018) ALT-803 Transiently Reduces Simian Immunodeficiency Virus Replication in the Absence of Antiretroviral Treatment. J Virol 92:
Park, Mi Ae; Jung, Ho Sun; Slukvin, Igor (2018) Genetic Engineering of Human Pluripotent Stem Cells Using PiggyBac Transposon System. Curr Protoc Stem Cell Biol 47:e63
Ellis, Amy; Balgeman, Alexis; Rodgers, Mark et al. (2017) Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques. Infect Immun 85:
Rodrigues, Michelle A (2017) Female Spider Monkeys (Ateles geoffroyi) Cope with Anthropogenic Disturbance Through Fission-Fusion Dynamics. Int J Primatol 38:838-855
Buechler, Connor R; Bailey, Adam L; Lauck, Michael et al. (2017) Genome Sequence of a Novel Kunsagivirus (Picornaviridae: Kunsagivirus) from a Wild Baboon (Papio cynocephalus). Genome Announc 5:
Wu, Hong; Whritenour, Jessica; Sanford, Jonathan C et al. (2017) Identification of MHC Haplotypes Associated with Drug-induced Hypersensitivity Reactions in Cynomolgus Monkeys. Toxicol Pathol 45:127-133
Shackman, A J; Fox, A S; Oler, J A et al. (2017) Heightened extended amygdala metabolism following threat characterizes the early phenotypic risk to develop anxiety-related psychopathology. Mol Psychiatry 22:724-732
Kalin, Ned H (2017) Mechanisms underlying the early risk to develop anxiety and depression: A translational approach. Eur Neuropsychopharmacol 27:543-553

Showing the most recent 10 out of 528 publications