| 61. |
- Marzec-Schmidt, Katarzyna, et al.
(författare)
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Artificial intelligence supports automated characterization of differentiated human pluripotent stem cells
- 2023
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Ingår i: Stem Cells. - : Oxford University Press. - 1066-5099 .- 1549-4918. ; 41:9, s. 850-861
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Tidskriftsartikel (refereegranskat)abstract
- Revolutionary advances in AI and deep learning in recent years have resulted in an upsurge of papers exploring applications within the biomedical field. Within stem cell research, promising results have been reported from analyses of microscopy images to e.g., distinguish between pluripotent stem cells and differentiated cell types derived from stem cells. In this work, we investigated the possibility of using a deep learning model to predict the differentiation stage of pluripotent stem cells undergoing differentiation towards hepatocytes, based on morphological features of cell cultures. We were able to achieve close to perfect classification of images from early and late time points during differentiation, and this aligned very well with the experimental validation of cell identity and function. Our results suggest that deep learning models can distinguish between different cell morphologies, and provide alternative means of semi-automated functional characterization of stem cell cultures.
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| 62. |
- McConkey, DJ, et al.
(författare)
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Signal transduction pathways in apoptosis
- 1996
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 14:6, s. 619-631
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Tidskriftsartikel (refereegranskat)
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| 63. |
- Miyake, Koichi, et al.
(författare)
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RPS19 Deficiency Leads to Reduced Proliferation and Increased Apoptosis but Does Not Affect Terminal Erythroid Differentiation in a Cell Line Model of Diamond-Blackfan Anemia
- 2008
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 26:2, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein (RP) S19 gene. It is not known how the RPS19 deficiency impairs erythropoiesis and proliferation of hematopoietic progenitors. To elucidate molecular mechanisms in RPS19 deficient DBA, we analyzed the effects of RPS19 deficiency on EPO induced signal transduction, cell cycle, and apoptosis in RPS19-deficient TF-1 cells. We did not find any abnormality in EPO induced signal transduction. However, RPS19 deficient-TF-1 cells showed G0/G1 arrest (82% vs 58%, p<0.05) together with accumulation of p21 and p27. The fraction of apoptotic cells detected by Annexin-V analysis also increased compared to control cells (13% vs 3.1%, p<0.05). Western blot analysis of apoptotic related proteins showed that the level of bcl-2 and Bad was decreased and Bax was increased in RPS19-deficient TF1 cells. Moreover, primary CD34 positive cells from DBA patients detected by Annexin-V analysis also generated a higher number of apoptotic cells compared to normal CD34 positive cells during in vitro culture (38% vs 8.9%, n=5, p<0.001). Finally, we show that while RPS19 silencing reduces EPO induced development of erythroid progenitors expressing Glycophorin A (GPA), RPS19 silencing in cells already expressing GPA does not affect GPA expression. These findings indicate that RPS19 deficiency causes apoptosis and accelerated loss of erythroid progenitors in RPS19 deficient DBA.
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| 64. |
- Miyake, Noriko, et al.
(författare)
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HOXB4-induced self-renewal of hematopoietic stem cells is significantly enhanced by p21 deficiency.
- 2006
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 24:3, s. 653-661
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Tidskriftsartikel (refereegranskat)abstract
- Enforced expression of the HOXB4 transcription factor and downregulation of p21Cip1/Waf (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21-deficient HSCs. HOXB4 was overexpressed in hematopoietic cells from wild-type (wt) and p21−/− mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4), or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture, the number of primary CFU-GM (colony-forming unit granulocyte-macrophage) colonies and the repopulating ability were significantly increased in MIGB4 p21−/− BM (p21B4) cells compared with MIGB4-transduced wt BM (wtB4) cells. To test proliferation of HSCs in vivo, we performed competitive repopulation experiments and obtained significantly higher long-term engraftment of expanded p21B4 cells compared with wtB4 cells. The 5-day expansion of p21B4 HSCs generated 100-fold higher numbers of competitive repopulating units compared with wtMIG and threefold higher numbers compared with wtB4. The findings demonstrate that increased expression of HOXB4, in combination with suppression of p21 expression, could be a useful strategy for effective and robust expansion of HSCs.
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| 65. |
- Mohsen-Kanson, T., et al.
(författare)
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Differentiation of Human Induced Pluripotent Stem Cells into Brown and White Adipocytes: Role of Pax3
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 32:6, s. 1459-1467
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Tidskriftsartikel (refereegranskat)abstract
- Identification of molecular mechanisms involved in generation of different types of adipocytes is progressing substantially in mice. However, much less is known regarding characterization of brown (BAP) and white adipocyte progenitors (WAPs) in humans, highlighting the need for an in vitro model of human adipocyte development. Here, we report a procedure to selectively derive BAP and WAPs from human-induced pluripotent stem cells. Molecular characterization of APs of both phenotypes revealed that BMP4, Hox8, Hoxc9, and HoxA5 genes were specifically expressed in WAPs, whereas expression of PRDM16, Dio2, and Pax3 marked BAPs. We focused on Pax3 and we showed that expression of this transcription factor was enriched in human perirenal white adipose tissue samples expressing UCP1 and in human classical brown fat. Finally, functional experiments indicated that Pax3 was a critical player of human AP fate as its ectopic expression led to convert WAPs into brown-like APs. Together, these data support a model in which Pax3 is a new marker of human BAPs and a molecular mediator of their fate. The findings of this study could lead to new anti-obesity therapies based on the recruitment of APs and constitute a platform for investigating in vitro the developmental origins of human white and brown adipocytes.
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| 66. |
- Moll, Guido, et al.
(författare)
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Are Therapeutic Human Mesenchymal Stromal Cells Compatible with Human Blood?
- 2012
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 30:7, s. 1565-1574
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Tidskriftsartikel (refereegranskat)abstract
- Multipotent mesenchymal stromal cells (MSCs) are tested in numerous clinical trials. Questions have been raised concerning fate and function of these therapeutic cells after systemic infusion. We therefore asked whether culture-expanded human MSCs elicit an innate immune attack, termed instant blood-mediated inflammatory reaction (IBMIR), which has previously been shown to compromise the survival and function of systemically infused islet cells and hepatocytes. We found that MSCs expressed hemostatic regulators similar to those produced by endothelial cells but displayed higher amounts of prothrombotic tissue/stromal factors on their surface, which triggered the IBMIR after blood exposure, as characterized by formation of blood activation markers. This process was dependent on the cell dose, the choice of MSC donor, and particularly the cell-passage number. Short-term expanded MSCs triggered only weak blood responses in vitro, whereas extended culture and coculture with activated lymphocytes increased their prothrombotic properties. After systemic infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D-dimer or acute-phase reactants with the currently applied dose of 1.0-3.0 x 10(6) cells per kilogram. Culture-expanded MSCs trigger the IBMIR in vitro and in vivo. Induction of IBMIR is dose-dependent and increases after prolonged ex vivo expansion. Currently applied doses of low-passage clinical-grade MSCs elicit only minor systemic effects, but higher cell doses and particularly higher passage cells should be handled with care. This deleterious reaction can compromise the survival, engraftment, and function of these therapeutic cells.
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| 67. |
- Moll, Guido, et al.
(författare)
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Do Cryopreserved Mesenchymal Stromal Cells Display Impaired Immunomodulatory and Therapeutic Properties?
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 32:9, s. 2430-2442
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Tidskriftsartikel (refereegranskat)abstract
- We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.
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| 68. |
- Mong, J, et al.
(författare)
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Transcription factor-induced lineage programming of noradrenaline and motor neurons from embryonic stem cells
- 2014
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:3, s. 609-622
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Tidskriftsartikel (refereegranskat)abstract
- An important goal in stem cell biology is to develop methods for efficient generation of clinically interesting cell types from relevant stem cell populations. This is particularly challenging for different types of neurons of the central nervous system where hundreds of distinct neuronal cell types are generated during embryonic development. We previously used a strategy based on forced transcription factor expression in embryonic stem cell-derived neural progenitors to generate specific types of neurons, including dopamine and serotonin neurons. Here, we extend these studies and show that noradrenergic neurons can also be generated from pluripotent embryonic stem cells by forced expression of the homeobox transcription factor Phox2b under the signaling influence of fibroblast growth factor 8 (FGF8) and bone morphogenetic proteins. In neural progenitors exposed to FGF8 and sonic hedgehog both Phox2b and the related Phox2a instead promoted the generation of neurons with the characteristics of mid- and hindbrain motor neurons. The efficient generation of these neuron types enabled a comprehensive genome-wide gene expression analysis that provided further validation of the identity of generated cells. Moreover, we also demonstrate that the generated cell types are amenable to drug testing in vitro and we show that variants of the differentiation protocols can be applied to cultures of human pluripotent stem cells for the generation of human noradrenergic and visceral motor neurons. Thus, these studies provide a basis for characterization of yet an additional highly clinically relevant neuronal cell type. Stem Cells 2014;32:609–622
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| 69. |
- Mononen, Mimmi, et al.
(författare)
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Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage
- 2020
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Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
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Tidskriftsartikel (refereegranskat)abstract
- A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.
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| 70. |
- Moody, Jennifer, et al.
(författare)
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Endoglin is not critical for hematopoietic stem cell engraftment and reconstitution but regulates adult erythroid development
- 2007
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 25:11, s. 2809-2819
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Tidskriftsartikel (refereegranskat)abstract
- Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC) However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.
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