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1.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360992

RESUMO

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.


Assuntos
Plaquetas/citologia , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Megacariócitos/citologia , Células Cultivadas , Meios de Cultura/química , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
2.
Theranostics ; 11(17): 8430-8447, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34373751

RESUMO

Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate. Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions. Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting. Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.

3.
Sci Rep ; 9(1): 7258, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076619

RESUMO

Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.


Assuntos
Expressão Gênica/fisiologia , Heparina/metabolismo , Células Estromais/metabolismo , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Soro/metabolismo , Transdução de Sinais/fisiologia , Cordão Umbilical/metabolismo
4.
Sci Rep ; 8(1): 12954, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154486

RESUMO

Application of in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is an increasingly popular strategy to transiently produce proteins as therapeutics in a tissue or organ of choice. Here, we focused on the skin and aimed to test if whole human skin tissue explant technology can be used to evaluate the expression efficacy of different IVT Interferon alpha (IFN-α) mRNA constructs in situ, after biolistic delivery. Skin explants were viable and intact for at least five days based on histologic analysis and TUNEL staining. Using GFP reporter mRNA formulations, we found mostly epidermal expression after biolistic delivery. Two out of five sequence-optimized IFN-α mRNA variants resulted in significantly improved IFN-α protein expression in human skin compared to native IFN-α mRNA transfection. IFN-α secretion analysis of the surrounding culture media confirmed these results. We provide a proof-of-concept that IFN-α mRNA delivery into intact human full thickness skin explants can be utilized to test mRNA sequence modifications ex vivo. This approach could be used to develop novel mRNA-based treatments of common epidermal skin conditions including non-melanoma skin cancer, where IFN-α protein therapy has previously shown a strong therapeutic effect.


Assuntos
Biolística , Epiderme , Expressão Gênica , Interferon-alfa , RNA Mensageiro , Neoplasias Cutâneas/terapia , Epiderme/metabolismo , Epiderme/patologia , Humanos , Interferon-alfa/biossíntese , Interferon-alfa/genética , Melanoma , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
5.
Theranostics ; 8(5): 1421-1434, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29507631

RESUMO

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation. Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC. Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells. Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.


Assuntos
Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , Tromboplastina/deficiência , Adulto , Animais , Coagulação Sanguínea , Contagem de Células , Tamanho Celular , Transplante de Células , Células Cultivadas , Feminino , Humanos , Imunomodulação , Masculino , Pessoa de Meia-Idade , Ratos , Fatores de Risco , Tromboembolia/etiologia , Tromboembolia/patologia , Tromboplastina/metabolismo , Adulto Jovem
6.
J Control Release ; 266: 87-99, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-28919557

RESUMO

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14+ dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy.


Assuntos
Alérgenos/administração & dosagem , Antígenos de Plantas/administração & dosagem , Células Dendríticas/imunologia , Lasers , Mananas/administração & dosagem , Pele/imunologia , Vacinação/métodos , Administração Cutânea , Animais , Ativação do Complemento , Feminino , Humanos , Imunoglobulina E/imunologia , Camundongos Endogâmicos BALB C , Porosidade , Células Th1/imunologia , Células Th17/imunologia
7.
PLoS One ; 8(11): e80483, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24303018

RESUMO

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.


Assuntos
Degeneração Retiniana/diagnóstico , Tomografia de Coerência Óptica , Animais , Animais Geneticamente Modificados , Morte Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Modelos Animais de Doenças , Células Ependimogliais/patologia , Células Ependimogliais/efeitos da radiação , Imuno-Histoquímica , Luz/efeitos adversos , Células Fotorreceptoras/patologia , Células Fotorreceptoras/efeitos da radiação , Retina/patologia , Retina/efeitos da radiação , Degeneração Retiniana/patologia , Neurônios Retinianos/patologia , Neurônios Retinianos/efeitos da radiação , Cicatrização , Peixe-Zebra
8.
PLoS One ; 7(1): e30365, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22291943

RESUMO

Fibroblast growth factors (Fgf) are secreted signaling molecules that have mitogenic, patterning, neurotrophic and angiogenic properties. Their importance during embryonic development in patterning and morphogenesis of the vertebrate eye is well known, but less is known about the role of Fgfs in the adult vertebrate retina. To address Fgf function in adult retina, we determined the spatial distribution of components of the Fgf signaling pathway in the adult zebrafish retina. We detected differential expression of Fgf receptors, ligands and downstream Fgf targets within specific retinal layers. Furthermore, we blocked Fgf signaling in the retina, by expressing a dominant negative variant of Fgf receptor 1 conditionally in transgenic animals. After blocking Fgf signaling we observe a fast and progressive photoreceptor degeneration and disorganization of retinal tissue, coupled with cell death in the outer nuclear layer. Following the degeneration of photoreceptors, a profound regeneration response is triggered that starts with proliferation in the inner nuclear layer. Ultimately, rod and cone photoreceptors are regenerated completely. Our study reveals the requirement of Fgf signaling to maintain photoreceptors and for proliferation during regeneration in the adult zebrafish retina.


Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/fisiologia , Peixe-Zebra , Fatores Etários , Animais , Animais Geneticamente Modificados , Morte Celular/genética , Morte Celular/fisiologia , Sobrevivência Celular/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Fotorreceptoras de Vertebrados/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Retina/citologia , Retina/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia
9.
Glia ; 58(11): 1345-63, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20607866

RESUMO

Adult telencephalic neurogenesis is a conserved trait of all vertebrates studied. It has been investigated in detail in rodents, but very little is known about the composition of neurogenic niches and the cellular nature of progenitors in nonmammalian vertebrates. To understand the components of the progenitor zones in the adult zebrafish telencephalon and the link between glial characteristics and progenitor state, we examined whether canonical glial markers are colocalized with proliferation markers. In the adult zebrafish telencephalon, we identify heterogeneous progenitors that reside in two distinct glial domains. We find that the glial composition of the progenitor zone is linked to its proliferative behavior. Analyzing both fast-cycling proliferating cells as well as slowly cycling progenitors, we find four distinct progenitor types characterized by differential expression of glial markers. Importantly, a significant proportion of progenitors do not display typical radial glia characteristics. By blocking or activating Fgf signaling by misexpression of a dominant negative Fgf-receptor 1 or Fgf8a, respectively, we find that ventral and dorsal progenitors in the telencephalon also differ in their requirement for Fgf signaling. Together with data on the expression of Fgf signaling components in the ventricular zone of the telencephalon, this suggests that Fgf signaling directly regulates proliferation of specific subsets of adult telencephalic progenitors in vivo. Taken together our results show that adult neural progenitor cells are heterogeneous with their respect to distribution into two distinct glial domains and their dependence upon Fgf signaling as a proliferative cue in the zebrafish telencephalon.


Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Células-Tronco/metabolismo , Telencéfalo/metabolismo , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Senescência Celular/fisiologia , Sinais (Psicologia) , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Neuroglia/citologia , Neurônios/citologia , Receptores de Fatores de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Especificidade da Espécie , Células-Tronco/citologia , Telencéfalo/citologia , Peixe-Zebra
10.
Gene Expr Patterns ; 7(3): 355-62, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16950663

RESUMO

Induction of the otic placode involves a number of regulatory interactions. Early studies revealed that the induction of this program is initiated by instructive signals from the mesendoderm as well as from the adjacent hindbrain. Further investigations on the molecular level identified in zebrafish Fgf3, Fgf8, Foxi1, Pax8, Dlx3b and Dlx4b genes as key players during the induction phase. Thereafter an increasing number of genes participates in the regulatory interactions finally resulting in a highly structured sensory organ. Based on data from zebrafish we selected medaka genes with presumptive functions during early ear development for an expression analysis. In addition we isolated Foxi1 and Dlx3b gene fragments from embryonic cDNA. Altogether we screened the spatio-temporal distribution of more than 20 representative marker genes for otic development in medaka embryos, with special emphasis on the early phases. Whereas the spatial distribution of these genes is largely conserved between medaka and zebrafish, our comparative analysis revealed several differences, in particular for the timing of expression.


Assuntos
Orelha/embriologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Oryzias/embriologia , Oryzias/genética , Animais , Animais Endogâmicos , Padronização Corporal/genética , DNA Complementar , Embrião não Mamífero/metabolismo , Fatores de Transcrição Forkhead/genética , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Fatores de Transcrição Box Pareados/genética , Fenótipo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
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