scispace - formally typeset
Search or ask a question
Author

Heide-Marie Binder

Bio: Heide-Marie Binder is an academic researcher from Paracelsus Private Medical University of Salzburg. The author has contributed to research in topics: Epidermolysis bullosa & Medicine. The author has an hindex of 3, co-authored 5 publications receiving 13 citations.

Papers
More filters
Journal ArticleDOI
TL;DR: It is demonstrated that nano‐sized EVs from therapy‐grade human placental‐expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology.
Abstract: Abstract Nanoparticles can acquire a plasma protein corona defining their biological identity. Corona functions were previously considered for cell‐derived extracellular vesicles (EVs). Here we demonstrate that nano‐sized EVs from therapy‐grade human placental‐expanded (PLX) stromal cells are surrounded by an imageable and functional protein corona when enriched with permissive technology. Scalable EV separation from cell‐secreted soluble factors via tangential flow‐filtration (TFF) and subtractive tandem mass‐tag (TMT) proteomics revealed significant enrichment of predominantly immunomodulatory and proangiogenic proteins. Western blot, calcein‐based flow cytometry, super‐resolution and electron microscopy verified EV identity. PLX‐EVs partly protected corona proteins from protease digestion. EVs significantly ameliorated human skin regeneration and angiogenesis in vivo, induced differential signalling in immune cells, and dose‐dependently inhibited T cell proliferation in vitro. Corona removal by size‐exclusion or ultracentrifugation abrogated angiogenesis. Re‐establishing an artificial corona by cloaking EVs with fluorescent albumin as a model protein or defined proangiogenic factors was depicted by super‐resolution microscopy, electron microscopy and zeta‐potential shift, and served as a proof‐of‐concept. Understanding EV corona formation will improve rational EV‐inspired nano‐therapy design.

40 citations

Journal ArticleDOI
TL;DR: In this paper, the authors investigated the effect of hypoxia on extracellular vesicles (EVs) in human induced pluripotent stem cells (iPSC) culture.
Abstract: Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.

13 citations

Journal ArticleDOI
TL;DR: Detailed on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1 editing strategy, associated with a significantly improved safety profile, and represent a further advancement in the field of traceless genome editing for genodermatoses.
Abstract: Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

11 citations

Journal ArticleDOI
TL;DR: Kocher et al. as discussed by the authors designed patient-specific Cas9-nuclease-and-nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression.

8 citations

Posted ContentDOI
17 Oct 2019-bioRxiv
TL;DR: Evidence is provided that EVs derived from therapeutic placental-expanded (PLX) stromal cells are potent inducers of angiogenesis and modulate immune cell proliferation in a dose-dependent manner, and indicates that the mode of PLX action involves an EV-mediated proangiogenic function and immune response modulation.
Abstract: Allogeneic regenerative cell therapy has shown surprising results despite lack of engraftment of the transplanted cells. Their efficacy was so far considered to be mostly due to secreted trophic factors. We hypothesized that extracellular vesicles (EVs) can also contribute to their mode of action. Here we provide evidence that EVs derived from therapeutic placental-expanded (PLX) stromal cells are potent inducers of angiogenesis and modulate immune cell proliferation in a dose-dependent manner. Crude EVs were enriched >100-fold from large volume PLX conditioned media via tangential flow filtration (TFF) as determined by tunable resistive pulse sensing (TRPS). Additional TFF purification was devised to separate EVs from cell-secreted soluble factors. EV identity was confirmed by western blot, calcein-based flow cytometry and electron microscopy. Surface marker profiling of tetraspanin-positive EVs identified expression of cell-and matrix-interacting adhesion molecules. Differential tandem mass tag proteomics comparing PLX-EVs to PLX-derived soluble factors revealed significant differential enrichment of 258 proteins in purified PLX-EVs involved in angiogenesis, cell movement and immune system signaling. At the functional level, PLX-EVs and cells inhibited T cell mitogenesis. PLX-EVs and soluble factors displayed dose-dependent proangiogenic potential by enhancing tube-like structure formation in vitro. Our findings indicate that the mode of PLX action involves an EV-mediated proangiogenic function and immune response modulation that may help explaining clinical efficacy beyond presence of the transplanted allogeneic cells.

7 citations


Cited by
More filters
01 Jan 2006
TL;DR: In this paper, a retroviral vector expressing LAMB3 cDNA (encoding LAM5-β3) was used to prepare genetically corrected cultured epidermal grafts, which were transplanted onto surgically prepared regions of the patient's legs.
Abstract: The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-β3–deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-β3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

55 citations

Journal ArticleDOI
TL;DR: This paper performed a systematic review using a text mining approach to assess adherence to the minimal information for studies of extracellular vesicles (MISEV) guidelines for assuring and improving EV research quality.

30 citations

Journal ArticleDOI
TL;DR: It is imperative to identify a reliable upscaling technique that can produce large quantities of EVs consistently and ideally, the produced EVs should also possess cargo with improved therapeutic potential.
Abstract: Extracellular vesicles (EVs) are minute vesicles with lipid bilayer membranes. EVs are secreted by cells for intercellular communication. Recently, EVs have received much attention, as they are rich in biological components such as nucleic acids, lipids, and proteins that play essential roles in tissue regeneration and disease modification. In addition, EVs can be developed as vaccines against cancer and infectious diseases, as the vesicle membrane has an abundance of antigenic determinants and virulent factors. EVs for therapeutic applications are typically collected from conditioned media of cultured cells. However, the number of EVs secreted by the cells is limited. Thus, it is critical to devise new strategies for the large-scale production of EVs. Here, we discussed the strategies utilized by researchers for the scalable production of EVs. Techniques such as bioreactors, mechanical stimulation, electrical stimulation, thermal stimulation, magnetic field stimulation, topographic clue, hypoxia, serum deprivation, pH modification, exposure to small molecules, exposure to nanoparticles, increasing the intracellular calcium concentration, and genetic modification have been used to improve the secretion of EVs by cultured cells. In addition, nitrogen cavitation, porous membrane extrusion, and sonication have been utilized to prepare EV-mimetic nanovesicles that share many characteristics with naturally secreted EVs. Apart from inducing EV production, these upscaling interventions have also been reported to modify the EVs’ cargo and thus their functionality and therapeutic potential. In summary, it is imperative to identify a reliable upscaling technique that can produce large quantities of EVs consistently. Ideally, the produced EVs should also possess cargo with improved therapeutic potential.

20 citations

Journal ArticleDOI
TL;DR: In this paper , the authors focus on the heterogeneity of extracellular vesicles derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions.
Abstract: Extracellular vesicles (EVs) are cell-derived membrane structures exerting major effects in physiological as well as pathological processes by functioning as vehicles for the delivery of biomolecules to their target cells. An increasing number of effects previously attributed to cell-based therapies have been recognized to be actually mediated by EVs derived from the respective cells, suggesting the administration of purified EVs instead of living cells for cell-based therapies. In this review, we focus on the heterogeneity of EVs derived from mesenchymal stem/stromal cells (MSC) and summarize upstream process parameters that crucially affect the resulting therapeutic properties and biological functions. Hereby, we discuss the effects of the cell source, medium composition, 3D culture, bioreactor culture and hypoxia. Furthermore, aspects of the isolation and storage strategies influences EVs are described. Conclusively, optimization of upstream process parameters should focus on controlling MSC-derived EV heterogeneity for specific therapeutic applications.

17 citations

Journal ArticleDOI
TL;DR: The roles and biogenesis of the EV corona, as well as the importance of controls to determine whether a biological effect is attributable to the internal EV cargo or to the corona associated with the EV exofacial surface are discussed.

15 citations