| 1. |
- de Peppo, Giuseppe Maria, 1981, et al.
(författare)
-
Human embryonic mesodermal progenitors highly resemble human mesenchymal stem cells and display high potential for tissue engineering applications.
- 2010
-
Ingår i: Tissue engineering. Part A. - : Mary Ann Liebert Inc. - 1937-335X .- 1937-3341. ; 16:7, s. 2161-82
-
Tidskriftsartikel (refereegranskat)abstract
- Adult stem cells, such as human mesenchymal stem cells (hMSCs), show limited proliferative capacity and, after long-term culture, lose their differentiation capacity and are therefore not an optimal cell source for tissue engineering. Human embryonic stem cells (hESCs) constitute an important new resource in this field, but one major drawback is the risk of tumor formation in the recipients. One alternative is to use progenitor cells derived from hESCs that are more lineage restricted but do not form teratomas. We have recently derived a cell line from hESCs denoted hESC-derived mesodermal progenitors (hES-MPs), and here, using genome-wide microarray analysis, we report that the process of hES-MPs derivation results in a significantly altered expression of hESC characteristic genes to an expression level highly similar to that of hMSCs. However, hES-MPs displayed a significantly higher proliferative capacity and longer telomeres. The hES-MPs also displayed lower expression of HLA class II proteins before and after interferon-gamma treatment, indicating that these cells may somewhat be immunoprivileged and potentially used for HLA-incompatible transplantation. The hES-MPs are thus an appealing alternative to hMSCs in tissue engineering applications and stem-cell-based therapies for mesodermal tissues.
|
|
| 2. |
- Gustavsson, Robert, 1987-
(författare)
-
Development of soft sensors for monitoring and control of bioprocesses
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the manufacture of bio-therapeutics the importance of a well-known process is key for a high product titer and low batch to batch variations. Soft sensors are based on the concept that online sensor signals can be used as inputs to mathematical models to derive new valuable process information. This information could then be used for better monitoring and control of the bioprocess.The aim of the present thesis has been to develop soft sensor solutions for upstream bioprocessing and demonstrate their usefulness in improving robustness and increase the batch-to-batch reproducibility in bioprocesses. The thesis reviews the potential and possibilities with soft sensors for use in production of bio-therapeutics to realize FDA´s process analytical technology (PAT) initiative. Modelling and hardware sensor alternatives which could be used in a soft sensor setup are described and critically analyzed. Different soft sensor approaches to control glucose feeding in fed-batch cultures of Escherichia coli are described. Measurements of metabolic fluxes and specific carbon dioxide production was used as control parameters to increase product yield and decrease the variability of produced recombinant proteins. Metabolic heat signals were used in uninduced cultures to estimate and control the specific growth rate at a desired level and thereby also estimate the biomass concentration online. The introduction of sequential filtering of the signal enabled this method to be used in a down-scaled system. The risk and high impact of contaminations in cell cultures are also described. An in situ microscope (ISM) was used as an online tool to estimate cell concentration and also to determine cell diameter size which enabled the detection of contaminant cells at an early stage.The work presented in this thesis supports the idea that soft sensors can be a useful tool in the strive towards robust and reliable bioprocesses, to ensure high product quality and increased economic profit.
|
|
| 3. |
- Adewumi, Oluseun, et al.
(författare)
-
Characterization of human embryonic stem cell lines by the International Stem Cell Initiative
- 2007
-
Ingår i: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 25:7, s. 803-816
-
Tidskriftsartikel (refereegranskat)abstract
- The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue- nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.
|
|
| 4. |
- Améen, Caroline, 1975, et al.
(författare)
-
Human embryonic stem cells: current technologies and emerging industrial applications.
- 2008
-
Ingår i: Critical reviews in oncology/hematology. - : Elsevier BV. - 1040-8428. ; 65:1, s. 54-80
-
Forskningsöversikt (refereegranskat)abstract
- The efficiency and accuracy of the drug development process is severely restricted by the lack of functional human cell systems. However, the successful derivation of pluripotent human embryonic stem (hES) cell lines in the late 1990s is expected to revolutionize biomedical research in many areas. Due to their growth capacity and unique developmental potential to differentiate into almost any cell type of the human body, hES cells have opened novel avenues both in basic and applied research as well as for therapeutic applications. In this review we describe, from an industrial perspective, the basic science that underlies the hES cell technology and discuss the current and future prospects for hES cells in novel and improved stem cell based applications for drug discovery, toxicity testing as well as regenerative medicine.
|
|
| 5. |
- Bigdeli, Narmin, 1974, et al.
(författare)
-
Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces.
- 2008
-
Ingår i: Journal of biotechnology. - : Elsevier BV. - 0168-1656. ; 133:1, s. 146-53
-
Tidskriftsartikel (refereegranskat)abstract
- Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigeltrade mark in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on Matrigel(TM). Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing.
|
|
| 6. |
- Bigdeli, Narmin, 1974, et al.
(författare)
-
Coculture of human embryonic stem cells and human articular chondrocytes results in significantly altered phenotype and improved chondrogenic differentiation.
- 2009
-
Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:8, s. 1812-21
-
Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem (hES) cells have been suggested as a cell source for the repair of cartilage lesions. Here we studied how coculture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers, and differentiation abilities of hES cells, with special regard to chondrogenic differentiation. Undifferentiated hES cells were cocultured with irradiated neonatal or adult articular chondrocytes in high-density pellet mass cultures for 14 days. Cocultured hES cells were then expanded on plastic and their differentiation potential toward the adipogenic, osteogenic, and chondrogenic lineages was compared with that of undifferentiated hES cells. The expression of different surface markers was investigated using flow cytometry and teratoma formation was studied using injection of the cells under the kidney capsule. Our results demonstrate that although hES cells have to be grown on Matrigel, the cocultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Coculture further resulted in a more homogenous pellet and significantly increased cartilage matrix production, both in high-density pellet mass cultures and hyaluronan-based scaffolds. Moreover, cocultured cells formed colonies in agarose suspension culture, also demonstrating differentiation toward chondroprogenitor cells, whereas no colonies were detected in the hES cell cultures. Coculture further resulted in a significantly decreased osteogenic potential. No teratoma formation was detected. Our results confirm the potential of the culture microenvironment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings.
|
|
| 7. |
- Bigdeli, Narmin, 1974, et al.
(författare)
-
Superior Osteogenic Capacity of Human Embryonic Stem Cells Adapted to Matrix-Free Growth Compared to Human Mesenchymal Stem Cells.
- 2010
-
Ingår i: Tissue engineering. Part A. - : Mary Ann Liebert Inc. - 1937-335X .- 1937-3341. ; 16:11, s. 3427-3440
-
Tidskriftsartikel (refereegranskat)abstract
- Human mesenchymal stem cells (hMSCs) represent a promising source of cells for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. An alternative is the use of human embryonic stem cells (hESCs), but labor-intensive expansion with the need for coating support limits their clinical use. We have previously derived a cell line from hESCs denoted matrix-free growth (MFG)-hESC that are independent of coating support for expansion, and we here compare its osteogenic capacity to that of hMSCs. Microarray analysis of hMSCs and MFG-hESCs revealed differential expression of genes involved in ossification. MFG-hESCs have significantly higher expression of secreted phosphoprotein 1 (SPP1) during osteogenic differentiation, whereas the opposite was true for alkaline phosphatase (ALPL), transforming growth factor, beta 1 (TGFB2), runt-related transcription factor 2 (RUNX2), and forkhead box C1 (FOXC1), as well as the activity of the ALPL enzyme, demonstrating that these two cell types differentiate into the osteogenic lineage using different signaling pathways. von Kossa staining, time-of-flight secondary ion mass spectrometry, and measurement of calcium and phosphate in the extracellular matrix demonstrated a superior ability of the MFG-hESCs to produce a mineralized matrix compared to hMSCs. The superior ability of the MFG-hESCs to form mineralized matrix compared to hMSCs demonstrates that MFG-hESCs are a promising alternative to the use of adult stem cells in future bone regenerative applications.
|
|
| 8. |
- Bisson, Isabelle, et al.
(författare)
-
Landscape of current and emerging cell therapy clinical trials in the UK: current status, comparison to global trends and future perspectives
- 2015
-
Ingår i: Regenerative Medicine. - : Future Medicine. - 1746-0751 .- 1746-076X. ; 10:2, s. 169-179
-
Tidskriftsartikel (refereegranskat)abstract
- Cell Therapy Clinical Trial and Preclinical Research databases have been established by the Cell Therapy Catapult to document current and future cell therapy clinical trials in the UK. We identified 41 ongoing trials in April 2014, an increase of seven trials from April 2013. In addition, we identified 45 late-stage preclinical research projects. The majority of the clinical trials are early phase, primarily led by academic groups. The leading therapeutic areas are cancer, cardiology and neurology. The trends in the UK are also seen globally. As the field matures, more later phase and commercial studies will emerge and the challenges will likely evolve into how to manufacture sufficient cell quantities, manage complex logistics for multi-center trials and control cost.
|
|
| 9. |
- Boreström, Cecilia, 1974, et al.
(författare)
-
Footprint-Free Human Induced Pluripotent Stem Cells From Articular Cartilage With Redifferentiation Capacity: A First Step Toward a Clinical-Grade Cell Source.
- 2014
-
Ingår i: Stem cells translational medicine. - : Oxford University Press (OUP). - 2157-6564 .- 2157-6580. ; 3:4, s. 433-447
-
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.
|
|
| 10. |
|
|
| 11. |
- Brechmann, Nils A., et al.
(författare)
-
Proof-of-Concept of a Novel Cell Separation Technology Using Magnetic Agarose-Based Beads
- 2022
-
Ingår i: MAGNETOCHEMISTRY. - : MDPI AG. - 2312-7481. ; 8:3, s. 34-
-
Tidskriftsartikel (refereegranskat)abstract
- The safety of the cells used for Advanced Therapy Medicinal Products is crucial for patients. Reliable methods for the cell purification are very important for the commercialization of those new therapies. With the large production scale envisioned for commercialization, the cell isolation methods need to be efficient, robust, operationally simple and generic while ensuring cell biological functionality and safety. In this study, we used high magnetized magnetic agarose-based beads conjugated with protein A to develop a new method for cell separation. A high separation efficiency of 91% yield and consistent isolation performances were demonstrated using population mixtures of human mesenchymal stem cells and HER2(+) SKBR3 cells (80:20, 70:30 and 30:70). Additionally, high robustness against mechanical stress and minimal unspecific binding obtained with the protein A base conjugated magnetic beads were significant advantages in comparison with the same magnetic microparticles where the antibodies were covalently conjugated. This study provided insights on features of large high magnetized microparticles, which is promising for the large-scale application of cell purification.
|
|
| 12. |
- de Peppo, Giuseppe Maria, 1981, et al.
(författare)
-
Human Embryonic Stem Cell-Derived Mesodermal Progenitors Display Substantially Increased Tissue Formation Compared to Human Mesenchymal Stem Cells Under Dynamic Culture Conditions in a Packed Bed/Column Bioreactor.
- 2013
-
Ingår i: Tissue engineering. Part A. - 1937-335X. ; 19:1-2, s. 175-87
-
Tidskriftsartikel (refereegranskat)abstract
- Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.
|
|
| 13. |
- de Peppo, Giuseppe Maria, et al.
(författare)
-
Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed Bed/Column bioreactor
- 2013
-
Ingår i: Tissue Engineering. Part A. - : Mary Ann Liebert Inc. - 1937-3341 .- 1937-335X. ; 19:1-2, s. 175-187
-
Tidskriftsartikel (refereegranskat)abstract
- Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.
|
|
| 14. |
- de Peppo, Giuseppe Maria, 1981, et al.
(författare)
-
Human Progenitor Cells for Bone Engineering Applications
- 2013
-
Ingår i: Current molecular medicine. - : Bentham Science Publishers. - 1566-5240 .- 1875-5666. ; 13:5, s. 723-734
-
Tidskriftsartikel (refereegranskat)abstract
- In this report, the authors review the human skeleton and the increasing burden of bone deficiencies, the limitations encountered with the current treatments and the opportunities provided by the emerging field of cell-based bone engineering. Special emphasis is placed on different sources of human progenitor cells, as well as their pros and cons in relation to their utilization for the large-scale construction of functional bone-engineered substitutes for clinical applications. It is concluded that, human pluripotent stem cells represent a valuable source for the derivation of progenitor cells, which combine the advantages of both embryonic and adult stem cells, and indeed display high potential for the construction of functional substitutes for bone replacement therapies.
|
|
| 15. |
- de Peppo, Giuseppe Maria, 1981, et al.
(författare)
-
Osteogenic Potential of Human Mesenchymal Stem Cells and Human Embryonic Stem Cell-Derived Mesodermal Progenitors: A Tissue Engineering Perspective.
- 2010
-
Ingår i: Tissue engineering. Part A. - : Mary Ann Liebert Inc. - 1937-335X .- 1937-3341. ; 16:11, s. 3413-3426
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Human mesenchymal stem cells (hMSCs) are promising candidates for bone engineering and regeneration with a considerable number of experimental successes reported over the last years. However, hMSCs show several limitations for tissue engineering applications, which can be overcome by using human embryonic stem cell-derived mesodermal progenitors (hES-MPs). The aim of this study was to investigate and compare the osteogenic differentiation potential of hMSCs and hES-MPs. Materials and Methods: The osteogenic differentiation and mineralization behavior of both cell types were evaluated at passage 5, 10, 15, and 20. Expression of COL1A1, RUNX2, OPN, and OC was evaluated by reverse transcription (RT)-polymerase chain reaction, whereas mineralization was examined by photospectrometry, von Kossa staining, and time-of-flight secondary ion mass spectrometry. The immunoprofile of both cell types was investigated by flow cytometry. Results: We demonstrated that, under proper stimulation, hES-MPs undergo osteogenic differentiation and exhibit significantly increased mineralization ability compared to hMSCs after protracted expansion. hES-MPs were also found to express lower amount of human leukocyte antigens class II proteins. Conclusions: The high osteogenic ability of hES-MPs, together with low expression of human leukocyte antigens class II, makes these cells an attractive alternative for bulk production of cells for bone engineering applications.
|
|
| 16. |
|
|
| 17. |
- Ellerström, Catharina, et al.
(författare)
-
Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation
- 2007
-
Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 25:7, s. 1690-1696
-
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.
|
|
| 18. |
- Englund, Mikael C. O., 1971, et al.
(författare)
-
The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.
- 2010
-
Ingår i: In vitro cellular & developmental biology. Animal. - : Springer Science and Business Media LLC. - 1543-706X .- 1071-2690. ; 46:3-4, s. 217-30
-
Tidskriftsartikel (refereegranskat)abstract
- This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.
|
|
| 19. |
- Hanson, Charles, 1958, et al.
(författare)
-
Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro.
- 2013
-
Ingår i: Acta ophthalmologica. - : Wiley. - 1755-3768 .- 1755-375X. ; 91:2, s. 127-130
-
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.
|
|
| 20. |
- Heins, Nico, et al.
(författare)
-
Clonal derivation and characterization of human embryonic stem cell lines.
- 2006
-
Ingår i: Journal of biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 122:4, s. 511-20
-
Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.
|
|
| 21. |
|
|
| 22. |
- Jergil, Måns, et al.
(författare)
-
Exploring Transcriptional Response toValproic Acid and Valproic Acid Analogs in Human Embryonic Stem Cells
-
Annan publikation (populärvet., debatt m.m.)abstract
- Developmental toxicity is a major concern for manufacturers of new pharmaceuticals,and current testing requires many laboratory animals. Human embryonic stem (hES)cells, potentially being close in function to cells in the developing embryo, mayprovide a technology for classification of candidate drugs in the early phase of toxicityevaluation. Altered gene expression in such system may be predictive of teratogenicproperties of a substance if important gene regulatory pathways are affected, and mayhence be used as appropriate endpoint. In the present study we used the pluripotenthES cell line SA002 (Cellartis AB), and microarrays to profile the response tovalproic acid (VPA), a known human teratogen causing increased risk of e.g. spinabifida and cognitive disorders in exposed embryos We also investigated three closelyrelated VPA analogs with differing in vivo teratogenicity in mice as well as histonedeacetylase (HDAC) inhibition, a proposed teratogenic mechanism of VPA. hEScells in an undifferentiated state were exposed for 24 h to either 1 mM VPA, 0.25mM or 0.5 mM (S)-2-pentyl-4-pentynoic acid a more potent teratogen and HDACinhibitor than VPA, 1 mM 3-propyl-heptanoic acid, a potent teratogen but not anHDAC inhibitor, 1 mM 2-ethyl-4-methyl-pentanoic acid, a non-teratogen and non-HDAC inhibitor, or 0.1% DMSO. Gene expression was subsequently profiled usingCodelink Human Whole Genome BioArrays. We found the HDAC inhibitors tostrongly deregulate largely the same genes. Further, a concordance of altered geneontology groups, predominantly neurogenic processes, was evident between all theteratogenic substances. Also, comparison with mouse ES cells showed an overlap ofderegulated genes as well as species specific gene to be deregulated.
|
|
| 23. |
- Kaneko, Tomoyuki, et al.
(författare)
-
On-chip in vitro cell-network pre-clinical cardiac toxicity using spatiotemporal human cardiomyocyte measurement on a chip
- 2014
-
Ingår i: Scientific Reports. - : Nature Publishing Group: Open Access Journals - Option B / Nature Publishing Group. - 2045-2322. ; 4:04670
-
Tidskriftsartikel (refereegranskat)abstract
- To overcome the limitations and misjudgments of conventional prediction of arrhythmic cardiotoxicity, we have developed an on-chip in vitro predictive cardiotoxicity assay using cardiomyocytes derived from human stem cells employing a constructive spatiotemporal two step measurement of fluctuation (short-term variability; STV) of cells repolarization and cell-to-cell conduction time, representing two origins of lethal arrhythmia. Temporal STV of field potential duration (FPD) showed a potential to predict the risks of lethal arrhythmia originated from repolarization dispersion for false negative compounds, which was not correctly predicted by conventional measurements using animal cells, even for non-QT prolonging clinical positive compounds. Spatial STV of conduction time delay also unveiled the proarrhythmic risk of asynchronous propagation in cell networks, whose risk cannot be correctly predicted by single-cell-based measurements, indicating the importance of the spatiotemporal fluctuation viewpoint of in vitro cell networks for precise prediction of lethal arrhythmia reaching clinical assessment such as thorough QT assay.
|
|
| 24. |
- Karlsson, Camilla, 1977, et al.
(författare)
-
Human embryonic stem cell-derived mesenchymal progenitors-Potential in regenerative medicine.
- 2009
-
Ingår i: Stem cell research. - : Elsevier BV. - 1876-7753 .- 1873-5061. ; 3:1, s. 39-50
-
Tidskriftsartikel (refereegranskat)abstract
- Tissue engineering and cell therapy require large-scale production of homogeneous populations of lineage-restricted progenitor cells that easily can be induced to differentiate into a specific tissue. We have developed straightforward protocols for the establishment of human embryonic stem (hES) cell-derived mesenchymal progenitor (hES-MP) cell lines. The reproducibility was proven by derivation of multiple hES-MP cell lines from 10 different hES cell lines. To illustrate clinical applicability, a xeno-free hES-MP cell line was also derived. None of the markers characteristic for undifferentiated hES cells were detected in the hES-MP cells. Instead, these cells were highly similar to mesenchymal stem cells with regard to morphology and expression of markers. The safety of hES-MP cells following transplantation was studied in severely combined immunodeficient (SCID) mice. The implanted hES-MP cells gave rise to homogeneous, well-differentiated tissues exclusively of mesenchymal origin and no teratoma formation was observed. These cells further have the potential to differentiate toward the osteogenic, adipogenic, and chondrogenic lineages in vitro. The possibility of easily and reproducibly generating highly expandable hES-MP cell lines from well-characterized hES cell lines with differentiation potential into several mesodermal tissues entails an enormous potential for the field of regenerative medicine.
|
|
| 25. |
- Larsson, D. G. Joakim, 1969, et al.
(författare)
-
Seasonal variations of vitelline envelope proteins, vitellogenin, and sex steroids in male and female eelpout (Zoarces viviparus).
- 2002
-
Ingår i: General and comparative endocrinology. - : Elsevier BV. - 0016-6480. ; 125:2, s. 184-96
-
Tidskriftsartikel (refereegranskat)abstract
- The seasonal variations of vitelline envelope proteins, vitellogenin (VTG), and reproductive steroids were investigated in feral male and female eelpout, Zoarces viviparus. 17beta-Estradiol was present in both sexes with a peak in prespawning fish of 2.6 ng/ml in males and 2.7 ng/ml in females. 11-Ketotestosterone peaked in June at 4.2 and 0.47 ng/ml in males and females, respectively. A surge of testosterone was seen in both sexes in August, just prior to spawning. All steroid levels were low during early pregnancy. The vitelline envelope of the eelpout is composed of two major and one minor protein with molecular weights of 50, 55, and 44 kDa, respectively. An antiserum raised against solubilized vitelline envelope from turbot (Scophthalmus maximus) cross-reacted strongly with the 50-kDa protein from the isolated vitelline envelope and a similar-sized protein in female plasma and plasma from estrogenized males. Interestingly, the 50-kDa protein was also present at low levels in males as demonstrated by ELISA and Western blotting. In males, the 50-kDa protein did not follow the seasonal changes in 17beta-estradiol, but instead showed an almost perfect negative correlation with water temperature. VTG was present in female plasma as shown by Western blotting, but VTG was not detectable in male plasma despite relatively high endogenous estrogen levels. This suggests that the VTG induction by estradiol may be modulated by other factors in the eelpout.
|
|
| 26. |
|
|
| 27. |
- Li, Ou, et al.
(författare)
-
Human Embryonic Stem Cell-Derived Mesenchymal Stroma Cells (hES-MSCs) Engraft In Vivo and Support Hematopoiesis without Suppressing Immune Function : Implications for Off-The Shelf ES-MSC Therapies
- 2013
-
Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 8:1
-
Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stroma cells (MSCs) have a high potential for novel cell therapy approaches in clinical transplantation. Commonly used bone marrow-derived MSCs (BM-MSCs), however, have a restricted proliferative capacity and cultures are difficult to standardize. Recently developed human embryonic stem cell-derived mesenchymal stroma cells (hES-MSCs) might represent an alternative and unlimited source of hMSCs. We therefore compared human ES-cell-derived MSCs (hES-MP002.5 cells) to normal human bone marrow-derived MSCs (BM-MSCs). hES-MP002.5 cells had lower yet reasonable CFU-F capacity compared with BM-MSC (8±3 versus 29±13 CFU-F per 100 cells). Both cell types showed similar immunophenotypic properties, i.e. cells were positive for CD105, CD73, CD166, HLA-ABC, CD44, CD146, CD90, and negative for CD45, CD34, CD14, CD31, CD117, CD19, CD 271, SSEA-4 and HLA-DR. hES-MP002.5 cells, like BM-MSCs, could be differentiated into adipocytes, osteoblasts and chondrocytes in vitro. Neither hES-MP002.5 cells nor BM-MSCs homed to the bone marrow of immune-deficient NSG mice following intravenous transplantation, whereas intra-femoral transplantation into NSG mice resulted in engraftment for both cell types. In vitro long-term culture-initiating cell assays and in vivo co-transplantation experiments with cord blood CD34+ hematopoietic cells demonstrated furthermore that hES-MP002.5 cells, like BM-MSCs, possess potent stroma support function. In contrast to BM-MSCs, however, hES-MP002.5 cells showed no or only little activity in mixed lymphocyte cultures and phytohemagglutinin (PHA) lymphocyte stimulation assays. In summary, ES-cell derived MSCs might be an attractive unlimited source for stroma transplantation approaches without suppressing immune function.
|
|
| 28. |
|
|
| 29. |
|
|
| 30. |
- Mount, Natalie M., et al.
(författare)
-
Cell-based therapy technology classifications and translational challenges
- 2015
-
Ingår i: Philosophical Transactions of the Royal Society of London. Biological Sciences. - : ROYAL SOC. - 0962-8436 .- 1471-2970. ; 370:1680, s. 20150017-
-
Forskningsöversikt (refereegranskat)abstract
- Cell therapies offer the promise of treating and altering the course of diseases which cannot be addressed adequately by existing pharmaceuticals. Cell therapies are a diverse group across cell types and therapeutic indications and have been an active area of research for many years but are now strongly emerging through translation and towards successful commercial development and patient access. In this article, we present a description of a classification of cell therapies on the basis of their underlying technologies rather than the more commonly used classification by cell type because the regulatory path and manufacturing solutions are often similar within a technology area due to the nature of the methods used. We analyse the progress of new cell therapies towards clinical translation, examine how they are addressing the clinical, regulatory, manufacturing and reimbursement requirements, describe some of the remaining challenges and provide perspectives on how the field may progress for the future.
|
|
| 31. |
- Poch, Christine M, et al.
(författare)
-
Migratory and anti-fibrotic programmes define the regenerative potential of human cardiac progenitors
- 2022
-
Ingår i: Nature Cell Biology. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1465-7392 .- 1476-4679.
-
Tidskriftsartikel (refereegranskat)abstract
- Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host–graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.
|
|
| 32. |
- Stenberg, Johan, et al.
(författare)
-
Sustained embryoid body formation and culture in a non-laborious three dimensional culture system for human embryonic stem cells
- 2011
-
Ingår i: Cytotechnology. - 0920-9069. ; 63:3, s. 227-237
-
Tidskriftsartikel (refereegranskat)abstract
- Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research. Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The agarose culture system was able to maintain expression of the three germ layers over 8 weeks of culture. All of the four hESC lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost effective, stable and non-laborious technique.
|
|
| 33. |
- Synnergren, Jane, 1967, et al.
(författare)
-
Transcriptional profiling of human embryonic stem cells differentiating to definitive and primitive endoderm and further toward the hepatic lineage.
- 2010
-
Ingår i: Stem cells and development. - : Mary Ann Liebert Inc. - 1557-8534 .- 1547-3287. ; 19:7, s. 961-78
-
Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESC) can differentiate into a variety of specialized cell types, and they constitute a useful model system to study embryonic development in vitro. In order to fully utilize the potential of these cells, the mechanisms that regulate the developmental processes of specific lineage differentiation need to be better defined. The aim of this study was to explore the molecular program involved in the differentiation of hESC toward definitive endoderm (DE) and further into the hepatic lineage, and to compare that with primitive endoderm (PrE) differentiation. To that end, we applied two protocols: a specific DE differentiation protocol and an intrinsic differentiation protocol that mainly mediates PrE formation. We collected hESC, hESC-derived DE, DE-derived hepatocyte-progenitors (DE-Prog), DE-derived hepatocyte-like cells (DE-Hep), and the corresponding PrE derivatives. The samples were analyzed using microarrays, and we identified sets of genes that were exclusively up-regulated in DE derivatives (compared to PrE derivatives) at discrete developmental stages. We also investigated known protein interactions among the set of up-regulated genes in DE-Hep. The results demonstrate important differences between DE and PrE differentiation on the transcriptional level. In particular, our results identify a unique molecular program, exclusively activated during development of DE and the subsequent differentiation of DE toward the hepatic lineage. We identified key genes and pathways of potential importance for future efforts to improve hepatic differentiation from hESC. These results reveal new opportunities for rational design of specific interventions with the purpose of generating enriched populations of DE derivatives, including functional hepatocytes.
|
|
| 34. |
- Williams, David J., et al.
(författare)
-
Comparability : Manufacturing, characterization and controls, report of a UK Regenerative Medicine Platform Pluripotent Stem Cell Platform Workshop, Trinity Hall, Cambridge, 14-15 September 2015
- 2016
-
Ingår i: Regenerative Medicine. - : Future Medicine Ltd. - 1746-0751 .- 1746-076X. ; 11:5, s. 483-492
-
Tidskriftsartikel (refereegranskat)abstract
- This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this 'may be difficult for cell-based medicinal products'. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.
|
|
