DNA aptamer assemblies as fibroblast growth factor mimics and their application in stem cell culture

Ueki, Ryosuke; Atsuta, Saki; Ueki, Ayaka; Hoshiyama, Junya; Li, Jingyue; Hayashi, Yohei; Sando, Shinsuke

doi:10.1039/c8cc08080a

Cited by 56 publications

(54 citation statements)

References 38 publications

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“…Recently, DNA aptamers have emerged as promising chemical entities for designing growth factor mimetics (10)(11)(12)(13). DNA aptamers can be synthesized chemically via well-established solid-phase synthesis methods, which have several advantages over biological production of recombinant proteins in terms of synthetic scalability and cost effectiveness (14,15).…”

Section: Introductionmentioning

confidence: 99%

A chemically unmodified agonistic DNA with growth factor functionality for in vivo therapeutic application

et al. 2020

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Although growth factors have great therapeutic potential because of their regenerative functions, they often have intrinsic drawbacks, such as low thermal stability and high production cost. Oligonucleotides have recently emerged as promising chemical entities for designing synthetic alternatives to growth factors. However, their applications in vivo have been recognized as a challenge because of their susceptibility to nucleases and limited distribution to a target tissue. Here, we present the first example of oligonucleotide-based growth factor mimetics that exerts therapeutic effects at a target tissue after systemic injection. The aptamer was designed to dimerize a growth factor receptor for its activation and mitigated the progression of Fas-induced fulminant hepatitis in a mouse model. This unprecedented functionality of the aptamer can be reasonably explained by its high nuclease stability and migration to the liver parenchyma. These mechanistic analyses provided insights for the successful application of aptamer-based receptor agonists.

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Section: Introductionmentioning

confidence: 99%

A chemically unmodified agonistic DNA with growth factor functionality for in vivo therapeutic application

et al. 2020

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“…This high abundance of bFGF levels in the CM of ARPE-19 cells is potentially useful as a stem cell maintenance medium, since sustained levels of bFGF improve the maintenance of human pluripotent and neural stem cells (Lotz et al, 2013). There have been persistent efforts to increase bFGF bioavailability (Han et al, 2017;Dvorak et al, 2018;Horighuchi et al, 2018;Titmarsh et al, 2017) in stem cell reserach because it has low thermal stability, being highly labile at 37°C (Levenstein et al, 2006;Furue et al, 2008), causing bFGF production efforts to suffer from batch-to-batch variations, and high costs of production (Ueki et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

Sharma

Kumar

Sharma

et al. 2019

Preprint

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During cell culture, trypsin, a serine protease, is applied to cells for 5-10 minutes to separate them from each other and from the underlying substratum so that they can be transferred to a different vessel, for re-plating, after growth medium containing 10 % serum has been added to the cells, in a well-known technique known as 'passaging'. The serum in the growth medium contains alpha-1 antitrypsin, which is a potent inhibitor of trypsin, elastase and other serine proteases.Although what is used is bovine serum in which levels of proteins could be different from levels seen in humans, normal human serum contains A1AT (> 1 mg/ml; > ~18 µmol/L) as well as trypsin itself (< 460 ng/ml, or ~0.02 µmol/L), with the former in a ~900-fold molar excess over the latter. Thus, it may be assumed there is also enough A1AT in the bovine serum added during passaging, to neutralize the trypsin (~100 μM) present in the small volume of trypsin-EDTA solution used to separate cells. What are the consequences of not adding serum, when growth medium is added, or of maintaining cells for a few tens of hours in the presence of trypsin, in a serum-free growth medium? What does such sustained exposure to trypsin during cell culture do to cells? More generally, what are the responses of cells within an organism to the balance of trypsin and A1AT in the serum that bathes them constantly? We know that excesses and deficiencies in the levels of either trypsin or A1AT are associated with disease. We know that cellular metabolism can be influenced through signaling involving protease activated membrane GPCR receptors (PAR1-4). In particular, we know of a receptor called PAR2, which is specifically activated by trypsin, expressed by cells at baseline levels, and upregulated through some feedback involving trypsin-activation. We also know that cells at sites of injury or inflammation produce and secrete trypsin, and that this trypsin can act locally upon cells in a variety of ways, all of which have probably not yet been elucidated. Here, we show that sustained exposure to trypsin induces cells to de-differentiate into a stem-like state. We show that if serum is either not added at all, or added and then washed away (after confluency is attained), during cell culture, all cells exposed to exogenously-added trypsin undergo changes in morphology, transcriptome, secretome, and developmental potential, and transition into a state of stemness, in minimal essential medium (MEM). Regardless of their origins, i.e., independent of whether they are derived from primary cultures, cell lines or cancer cell lines, and regardless of the original cell type used, exposure to trypsin (~10 µM; ~250 µg/ml) at a concentration 10-fold lower than that used to separate cells during passaging (~100 μM), over a period of 24-48 hours, causes cells to (1) become rounded, (2) cluster together, (3) get arrested in the G0/G1 stage of the cell cycle, (4) display increased presence of 5-hydroxymethyl cytosine in their nuclei (indicative of reprogramming), (5) display increased...

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“…[181][182][183] Some examples of these mimetic aptamers that have been developed so far include: a divalent agonist of VEGF receptor-2, which could selectively bind, dimerize, and activate this receptor; subsequent activation of downstream Akt pathway led to upregulation of endothelial nitric oxide synthase and formation of endothelial cell capillary tubes; [181] an HGF mimetic which, as a dimer, could induce Met receptor activation at nanomolar potency, reproducing HGF-induced cell responses such as migration and proliferation; [182] and a 76-mer single-stranded DNA sequence that could support the selfrenewal and pluripotency characteristics of induced pluripotent stem cells, replacing FGF-2 in culture conditions. [183] As has been shown in recent years, aptamers have increasingly appeared as valid options to develop biomaterials with not only increased affinity for GFs in general, but also specificity and dynamic programmability. These properties allow an evergreater degree of control over biomaterial biofunctionalization and, therefore, a greater precision in eliciting determined cellular responses.…”

Section: Selective Ligands For Gf Capturementioning

confidence: 99%

Biomaterials for Sequestration of Growth Factors and Modulation of Cell Behavior

Teixeira

Domingues

Shevchuk

et al. 2020

Adv Funct Materials

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Growth factors (GFs) are proteins secreted by cells that regulate a variety of biological processes. Although they have long been proposed as potent therapeutic agents, their administration in a soluble form has proven costly and ineffective due to their short half-lives in biological environments. Biomaterial-based approaches are increasingly sought as alternatives to improve the efficacy or, ideally, replace the need for exogenous administration of GFs in regenerative medicine strategies. The means by which these systems evolve from biomaterials for conventional controlled release of GFs to the recent extracellular matrix (ECM)-inspired approaches for sequestering these labile molecules and regulating their spatiotemporal activity and presentation are reviewed. Focus is placed on biomaterials functionalized either with ECM components, which show promiscuous GF binding, or with targeted GF ligands (antibodies, aptamers, or peptides). The potential of synthetic platforms with abiotic affinity as cost-effective alternatives to the current biological ligands is also discussed. Overall, the various GF sequestering systems developed so far have remarkably improved the activity of GFs at reduced doses and, in some cases, completely avoided the need for their exogenous administration to guide cell fates. These bioinspired concepts thus enable the rational exploration of the full therapeutic potential of GFs in regenerative medicine.

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DNA aptamer assemblies as fibroblast growth factor mimics and their application in stem cell culture

Abstract: A 76-mer single-stranded DNA acts as functional mimic of basic fibroblast growth factor (bFGF), one of the essential factors for stem cell culture.

Cited by 56 publications

References 38 publications

A chemically unmodified agonistic DNA with growth factor functionality for in vivo therapeutic application

A chemically unmodified agonistic DNA with growth factor functionality for in vivo therapeutic application

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

Biomaterials for Sequestration of Growth Factors and Modulation of Cell Behavior

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