Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Megakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 × 10 ⁷ MKs per 50 ml medium. Laminin-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion. In vitro generated MKs represent a promising alternative to donor PLTs and open the possibility for the development of innovative MK-based cell therapies.

Related collections

Most cited references 38

Record: found
Abstract: found
Article: not found

Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells.

Meng Zhao, John M Perry, Heather Marshall … (2014)

Multiple bone marrow stromal cell types have been identified as hematopoietic stem cell (HSC)-regulating niche cells. However, whether HSC progeny can serve directly as HSC niche cells has not previously been shown. Here we report a dichotomous role of megakaryocytes (MKs) in both maintaining HSC quiescence during homeostasis and promoting HSC regeneration after chemotherapeutic stress. We show that MKs are physically associated with HSCs in the bone marrow of mice and that MK ablation led to activation of quiescent HSCs and increased HSC proliferation. RNA sequencing (RNA-seq) analysis revealed that transforming growth factor β1 (encoded by Tgfb1) is expressed at higher levels in MKs as compared to other stromal niche cells. MK ablation led to reduced levels of biologically active TGF-β1 protein in the bone marrow and nuclear-localized phosphorylated SMAD2/3 (pSMAD2/3) in HSCs, suggesting that MKs maintain HSC quiescence through TGF-β-SMAD signaling. Indeed, TGF-β1 injection into mice in which MKs had been ablated restored HSC quiescence, and conditional deletion of Tgfb1 in MKs increased HSC activation and proliferation. These data demonstrate that TGF-β1 is a dominant signal emanating from MKs that maintains HSC quiescence. However, under conditions of chemotherapeutic challenge, MK ablation resulted in a severe defect in HSC expansion. In response to stress, fibroblast growth factor 1 (FGF1) signaling from MKs transiently dominates over TGF-β inhibitory signaling to stimulate HSC expansion. Overall, these observations demonstrate that MKs serve as HSC-derived niche cells to dynamically regulate HSC function.

0 comments Cited 241 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Functional diversity of laminins.

Anna Domogatskaya, Sergey Rodin, Karl Tryggvason (2012)

Laminins are a large family of conserved, multidomain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behavior of associated cells, such as adhesion, differentiation, migration, phenotype stability, and resistance to anoikis. In lower organisms such as Hydra there is only one isoform of laminin, but higher organisms have at least 16 trimeric isoforms with varying degrees of cell/tissue specificity. In vitro protein and cell culture studies, gene manipulation in animals, and laminin gene mutations in human diseases have provided insight into the specific functions of some laminins, but the biological roles of many isoforms are still largely unexplored, mainly owing to difficulties in isolating them in pure form from tissues or cells. In this review, we elucidate the evolution of laminins, describe their molecular complexity, and explore the current knowledge of their diversity and functional aspects, including laminin-mediated signaling via membrane receptors, in vitro cell biology, and involvement in various tissues gained from animal model and human disease studies. The potential use of laminins in cell biology research and biotechnology is discussed.

0 comments Cited 151 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

Masashi Yamada, Maria Hayashi, T. Tsuji … (2006)

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.

0 comments Cited 122 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Contributors

Constança Figueiredo: Figueiredo.Constanca@mh-hannover.de

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 5 July 2018

Publication date PMC-release: 5 July 2018

Publication date Collection: 2018

Volume: 8

Electronic Location Identifier: 10146

Affiliations

[1 ]ISNI 0000 0000 9529 9877, GRID grid.10423.34, Institute for Transfusion Medicine, , Hannover Medical School, ; Hannover, 30625 Germany

[2 ]GRID grid.7490.a, Department of Vaccinology and Applied Microbiology, , Helmholtz Centre for Infection Research, ; Braunschweig, 38124 Germany

[3 ]ISNI 0000 0000 9529 9877, GRID grid.10423.34, Institute for Biophysical Chemistry, , Hannover Medical School, ; Hannover, 30625 Germany

[4 ]Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover, 30625 Germany

[5 ]ISNI 0000 0000 9529 9877, GRID grid.10423.34, REBIRTH Cluster of Excellence, , Hannover Medical School, ; Hannover, 30625 Germany

Author information

Robert Zweigerdt http://orcid.org/0000-0002-4656-0770

Article

Publisher ID: 28459

DOI: 10.1038/s41598-018-28459-x

PMC ID: 6033877

PubMed ID: 29977045

SO-VID: d7637b8f-bfc1-409c-bfd0-07922c9b6e64

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 2 March 2018

Date accepted : 21 June 2018

Funding

Funded by: FundRef https://doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft (German Research Foundation);

Award ID: EXC32 Unit 6.3

Award ID: EXC62 Unit 6.3

Award Recipient : Rainer Blasczyk Constança Figueiredo

Custom metadata

ScienceOpen disciplines: Uncategorized

Data availability:

ScienceOpen disciplines: Uncategorized

Comments

Comment on this article

scite_

Cited by 13

See all cited by

Most referenced authors 462

See all reference authors

Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors

Read this article at

Abstract

Related collections

SDG: 12: Responsible Consumption and Production

Most cited references 38

Megakaryocytes maintain homeostatic quiescence and promote post-injury regeneration of hematopoietic stem cells.

Functional diversity of laminins.

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

Author and article information

Contributors

Journal

Affiliations

Author information

Article

History

Funding

Categories

Custom metadata

Comments

Comment on this article

Similar content 456

Cited by 13

Most referenced authors 462