WiCell Research Institute

Madison, WI, United States

WiCell Research Institute

Madison, WI, United States
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Vodyanik M.A.,University of Wisconsin - Madison | Yu J.,University of Wisconsin - Madison | Zhang X.,WiCell Research Institute | Tian S.,Morgridge Institute for Research | And 5 more authors.
Cell Stem Cell | Year: 2010

Among the three embryonic germ layers, the mesoderm is a major source of the mesenchymal precursors giving rise to skeletal and connective tissues, but these precursors have not previously been identified and characterized. Using human embryonic stem cells directed toward mesendodermal differentiation, we show that mesenchymal stem/stromal cells (MSCs) originate from a population of mesodermal cells identified by expression of apelin receptor. In semisolid medium, these precursors form FGF2-dependent compact spheroid colonies containing mesenchymal cells with a transcriptional profile representative of mesoderm-derived embryonic mesenchyme. When transferred to adherent cultures, individual colonies give rise to MSC lines with chondro-, osteo-, and adipogenic differentiation potentials. Although the MSC lines lacked endothelial potential, endothelial cells could be derived from the mesenchymal colonies, suggesting that, similar to hematopoietic cells, MSCs arise from precursors with angiogenic potential. Together, these studies identified a common precursor of mesenchymal and endothelial cells, mesenchymoangioblast, as the source of mesoderm-derived MSCs. © 2010 Elsevier Inc.


Trademark
Wisconsin Alumni Research Foundation and WiCell Research Institute | Date: 2013-07-23

Biological tissues, namely, stem cells for scientific use. Biological tissues, namely, stem cells for medical use. Scientific research support services, namely, providing telephonic and online information about the culture and use of stem cells for stem cell researchers using stem cells.


Trademark
Wisconsin Alumni Research Foundation and WiCell Research Institute | Date: 2013-04-16

Biological tissues, namely, cells for scientific use. Biological tissues, namely, cells for medical use. Educational services, namely, providing classes, seminars and laboratory instruction in the field of culturing and using cells; training services in the field of culturing and using cells. Scientific research support services, namely, providing telephonic and online information about the culture and use of cells for individuals using cells.


Hu K.,University of Wisconsin - Madison | Yu J.,University of Wisconsin - Madison | Suknuntha K.,University of Wisconsin - Madison | Tian S.,Morgridge Institute for Research | And 5 more authors.
Blood | Year: 2011

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using nonintegrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Bloodderived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9, bloodderived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML).CMLiPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.


Azarin S.M.,University of Wisconsin - Madison | Azarin S.M.,WiCell Research Institute | Lian X.,University of Wisconsin - Madison | Lian X.,WiCell Research Institute | And 5 more authors.
Biomaterials | Year: 2012

Intercellular interactions in the cell microenvironment play a critical role in determining cell fate, but the effects of these interactions on pathways governing human embryonic stem cell (hESC) behavior have not been fully elucidated. We and others have previously reported that 3-D culture of hESCs affects cell fates, including self-renewal and differentiation to a variety of lineages. Here we have used a microwell culture system that produces 3-D colonies of uniform size and shape to provide insight into the effect of modulating cell-cell contact on canonical Wnt/β-catenin signaling in hESCs. Canonical Wnt signaling has been implicated in both self-renewal and differentiation of hESCs, and competition for β-catenin between the Wnt pathway and cadherin-mediated cell-cell interactions impacts various developmental processes, including the epithelial-mesenchymal transition. Our results showed that hESCs cultured in 3-D microwells exhibited higher E-cadherin expression than cells on 2-D substrates. The increase in E-cadherin expression in microwells was accompanied by a downregulation of Wnt signaling, as evidenced by the lack of nuclear β-catenin and downregulation of Wnt target genes. Despite this reduction in Wnt signaling in microwell cultures, embryoid bodies (EBs) formed from hESCs cultured in microwells exhibited higher levels of Wnt signaling than EBs from hESCs cultured on 2-D substrates. Furthermore, the Wnt-positive cells within EBs showed upregulation of genes associated with cardiogenesis. These results demonstrate that modulation of intercellular interactions impacts Wnt/β-catenin signaling in hESCs. © 2011 Elsevier Ltd.


Klim J.R.,University of Wisconsin - Madison | Li L.,University of Wisconsin - Madison | Wrighton P.J.,University of Wisconsin - Madison | Piekarczyk M.S.,WiCell Research Institute | Kiessling L.L.,University of Wisconsin - Madison
Nature Methods | Year: 2010

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells. © 2010 Nature America, Inc. All rights reserved.


Xu X.,University of Wisconsin - Madison | Xu X.,WiCell Research Institute | Browning V.L.,University of Wisconsin - Madison | Odorico J.S.,University of Wisconsin - Madison | Odorico J.S.,WiCell Research Institute
Mechanisms of Development | Year: 2011

The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3-4days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21-28days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1 + clusters are formed. Almost all cells in PDX1 + clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7-14days, then the number of insulin + cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination. © 2011 Elsevier Ireland Ltd.


Shevde N.K.,WiCell Research Institute | Mael A.A.,WiCell Research Institute
Methods in Molecular Biology | Year: 2013

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels, under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm, mesoderm, and endoderm. In this chapter, we describe techniques for generation of EBs from human pluripotent stem cells. Once formed, the EBs can then be dissociated using specific enzymes to acquire a single cell population that has the potential to differentiate into cells of all three germ layers. This population can then be cultured in specialized conditions to obtain progenitor cells of specific lineages. Pure populations of progenitor cells generated on a large scale basis can be used for research, drug discovery/development, and cellular transplantation therapy. © 2013 Springer Science+Business Media, LLC.


Azarin S.M.,University of Wisconsin - Madison | Azarin S.M.,WiCell Research Institute | Palecek S.P.,University of Wisconsin - Madison | Palecek S.P.,WiCell Research Institute
Biochemical Engineering Journal | Year: 2010

The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and substrates, monitoring spontaneous differentiation and heterogeneity in the cultures, and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. © 2009 Elsevier B.V. All rights reserved.


Patent
WiCell Research Institute | Date: 2012-02-17

The invention relates to methods for culturing human embryonic stem cells by culturing the stem cells in an environment essentially free of mammalian fetal serum and in a stem cell culture medium including amino acids, vitamins, salts, minerals, transferrin, insulin, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer the medium. Also disclosed are compositions capable of supporting the culture and proliferation of human embryonic stem cells without the need for feeder cells or for exposure of the medium to feeder cells.

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