| 1. |
- Boreström, Cecilia, 1974, et al.
(författare)
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Footprint-Free Human Induced Pluripotent Stem Cells From Articular Cartilage With Redifferentiation Capacity: A First Step Toward a Clinical-Grade Cell Source.
- 2014
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Ingår i: Stem cells translational medicine. - : Oxford University Press (OUP). - 2157-6564 .- 2157-6580. ; 3:4, s. 433-447
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Tidskriftsartikel (refereegranskat)abstract
- Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint-free iPSCs (no genome-sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix-forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte-derived iPSCs can be redifferentiated in vitro into cartilage matrix-producing cells better than fibroblast-derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte-derived iPSCs a promising candidate universal cell source for ACI. Whole-genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA-based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical-grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.
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| 2. |
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| 3. |
- Ellerström, Catharina, et al.
(författare)
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Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation
- 2007
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 25:7, s. 1690-1696
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Tidskriftsartikel (refereegranskat)abstract
- Traditionally, human embryonic stem cells (hESCs) are propagated by mechanical dissection or enzymatic dissociation into clusters of cells. To facilitate up-scaling and the use of hESC in various experimental manipulations, such as fluorescence-activated cell sorting, electroporation, and clonal selection, it is important to develop new, stable culture systems based on single-cell enzymatic propagation. Here, we show that hESCs, which were derived and passaged by mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. As an indication of the stability of this culture system, we demonstrate that hESCs can be maintained in an undifferentiated, pluripotent, and genetically normal state for up to 40 enzymatic passages. We also demonstrate that a recombinant trypsin preparation increases clonal survival compared with porcine trypsin. Finally, we show that human foreskin fibroblast feeders are superior to the commonly used mouse embryonic fibroblast feeders in terms of their ability to prevent spontaneous differentiation after single-cell passaging. Importantly, the culture system is widely applicable and should therefore be of general use to facilitate reliable large-scale cultivation of hESCs, as well as their use in various experimental manipulations.
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| 4. |
- Ellerström, Catharina
(författare)
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Gene expression during hard tissue formation
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The purpose of this thesis was to study the formation of the extracellular matrix (ECM) of bone and dentin during development. Molecular techniques, such as in situ hybridization and RT-PCR, were used to investigate the occurrence of specific gene products in formative cells. The fibrillar collagen type I is the major extracellular component in both bone and dentin. According to Northern blots, the [alpha]1(I) mRNA chain is composed of two variants. The complete sequences of the [alpha]1(I) chains were established and it was found that these differ in the untranslated regions only. The expression patterns of the two variants were compared in the hard tissue forming cells of growing rat maxilla and they were found to be expressed at all developmental stages and in all different cell types studied. The mRNA expression of the fibril collagens, types I, II, III, V and XI was compared during formation of the premaxillary-maxillary suture region, an area of intramembranous bone formation. A specific temporospacial expression pattern was found, which could be correlated to collagen fiber construction and tissue segregation. Type III mRNA was expressed in the soft tissues of the suture and periosteum, types V and XI mRNA occurred only transiently in young osteoblasts at the border between soft and hard tissue, while type I transcript was detected in all osteoblasts, but with different signal strength depending on the localization of the cell. Type II mRNA was not detected in the suture region. In contrast, no sutures were formed and no collagen type III mRNA expression took place during the growth of the alveolar bone. Instead, collagen H mRNA was expressed in islands of cells, which later developed into cartilage domains, and which were interspersed between osteoblasts synthesizing collagen type I mRNA, and bone trabeculae. It is proposed that collagen type II and cartilage are instrumental for bone growth and allow bone expansion, functionally replacing collagen type III and sutures. One of the major non-collagenous extracellular matrix components in both bone and teeth is osteonectin (ON). The sequence of the rat ON mRNA was established and the expression pattern of its mRNA was mapped and found to be similar to the pattern obtained with collagen [alpha]1(I). It seems likely that ON is synthesized concomitantly with the fibrillar collagen proteins and is a component of the ECM. Ameloblasts, associated with mineralizing enamel did not express ON mRNA, thus presence of ON mRNA and the deposition of mineralizing enamel are not correlated. Fibronectin (FN) is also produced by osteoblasts and odontoblasts and is known to be involved in cell attachment but can also bind to e.g. collagen. At three different positions, the transcript can e alternatively spliced, presumably to allow for variable functions of FN. The splicing pattern in bone and teeth cells was investigated. Osteoblasts omitted and odontoblasts used exon EM, a difference that could be related to specific modes of cell-matrix organization.
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| 5. |
- Hanson, Charles, 1958, et al.
(författare)
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Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro.
- 2013
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Ingår i: Acta ophthalmologica. - : Wiley. - 1755-3768 .- 1755-375X. ; 91:2, s. 127-130
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells. Methods: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. Results: The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3days after transplantation, and after 6days, the cells were expressing both PAX6 and CK3. Conclusion: This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.
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