Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45 ؉ CD19 ؉ CD10 ؉ and were positive for transcripts Pax5, IL7␣R, -like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45 ؉ CD19 ؉ cells also exhibited multiple genomic D-J H rearrangements, which supports a pre-B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage. (Blood. 2011;117(15): 4008-4011) IntroductionReprogramming of adult human somatic cells toward induced pluripotent stem cells 1-3 is a groundbreaking technology that opens the way for the development of novel diagnostics and therapeutics. Although traditionally human embryonic stem (hES) cells have been used as a convenient tool with which to understand hematopoietic and lymphoid development in health and disease, the usefulness of human induced pluripotent stem (iPS) cells for this purpose has recently been questioned. 4 For hES cells, a limited number of studies have derived B cells 5 and T cells, 6,7 with some reports suggesting why this might be difficult to achieve, 8 and which has not been described thus far for B cells from human iPS cells. Development of lymphoid progenitors from such pluripotent sources is of particular interest because it will help to understand better the early stages of B-cell development, which is currently not well understood. 9 This includes which B cells are formed and when and how these lineages relate to the formation of both normal and leukemic progenitors during development. 10,11 Methods Derivation, identification, and maintenance of human iPS cell lines iPS cells were generated essentially as decided by Takahashi et al, 1 with changes highlighted herein. pMX vectors hOct4, hSox2, and hKlf4 were purchased from Addgene and viral supernantants for each transgene produced in pantropic PLATGP packaging cells (Cell Biolabs). Transduced Normal Human Dermal Fibroblasts (NHDFs; Lonza Biologics) were cocultured with mitomycin C-treated mouse embryonic fibroblasts in modified embryonic stem cell media (Dulbecco modified Eagle medium/ F12, 20% [v/v] Knock Out Serum Replacement, 10 ng/mL basic fibroblast growth factor, 2mM sodium pyruvate, 1ϫ nonessential amino acids [Invitrogen], and 1M mercaptoethanol [Sigma-Aldrich]). Cultures were routinely maintained in 5% CO 2 and 5% oxygen with half-media changes every 2 days, for up to 34 days. iPS colonies were picked and expanded before adaptation to feeder independent culture as describ...
The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell lines and primary cells to lambdaDNA standards was also conducted. An effective working range of the assay within 3D constructs was shown up to 2.5 x 10(5) cells ml(-1). From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate standard to use for the assay was from the cell type being examined.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.