Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate

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Abstract

Background

Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology.

Methods

Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed.

Results

Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na ⁺ were elevated in pHPL preparations, K ⁺ and Fe ³⁺ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions.

Conclusion

All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.

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A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells.

Hitoshi Niwa, Kazuya Ogawa, Daisuke Shimosato … (2009)

The cytokine leukaemia inhibitory factor (LIF) integrates signals into mouse embryonic stem (ES) cells to maintain pluripotency. Although the Jak-Stat3 pathway is essential and sufficient to mediate LIF signals, it is still unclear how these signals are linked to the core circuitry of pluripotency-associated transcription factors, consisting of Oct3/4 (also called Pou5f1), Sox2 and Nanog. Here we show that two LIF signalling pathways are each connected to the core circuitry via different transcription factors. In mouse ES cells, Klf4 is mainly activated by the Jak-Stat3 pathway and preferentially activates Sox2, whereas Tbx3 is preferentially regulated by the phosphatidylinositol-3-OH kinase-Akt and mitogen-activated protein kinase pathways and predominantly stimulates Nanog. In the absence of LIF, artificial expression of Klf4 or Tbx3 is sufficient to maintain pluripotency while maintaining the expression of Oct3/4. Notably, overexpression of Nanog supports LIF-independent self-renewal of mouse ES cells in the absence of Klf4 and Tbx3 activity. Therefore, Klf4 and Tbx3 are involved in mediating LIF signalling to the core circuitry but are not directly associated with the maintenance of pluripotency, because ES cells keep pluripotency without their expression in the particular context.

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Single Cell RNA-Sequencing of Pluripotent States Unlocks Modular Transcriptional Variation

Aleksandra A. Kolodziejczyk, Jong Kim, Jason C.H. Tsang … (2015)

Summary Embryonic stem cell (ESC) culture conditions are important for maintaining long-term self-renewal, and they influence cellular pluripotency state. Here, we report single cell RNA-sequencing of mESCs cultured in three different conditions: serum, 2i, and the alternative ground state a2i. We find that the cellular transcriptomes of cells grown in these conditions are distinct, with 2i being the most similar to blastocyst cells and including a subpopulation resembling the two-cell embryo state. Overall levels of intercellular gene expression heterogeneity are comparable across the three conditions. However, this masks variable expression of pluripotency genes in serum cells and homogeneous expression in 2i and a2i cells. Additionally, genes related to the cell cycle are more variably expressed in the 2i and a2i conditions. Mining of our dataset for correlations in gene expression allowed us to identify additional components of the pluripotency network, including Ptma and Zfp640, illustrating its value as a resource for future discovery.

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Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released.

Joseph E. Italiano, Jennifer Richardson, Sunita Patel-Hett … (2008)

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro- and antiangiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules. These observations motivated the hypothesis that distinct populations of alpha-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH(2)) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH(2)) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis.

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Author and article information

Contributors

Katharina Schallmoser:

ORCID: http://orcid.org/0000-0002-7204-0058

k.schallmoser@salk.at

Journal

Journal ID (nlm-ta): J Transl Med

Journal ID (iso-abbrev): J Transl Med

Title: Journal of Translational Medicine

Publisher: BioMed Central (London )

ISSN (Electronic): 1479-5876

Publication date (Electronic): 30 December 2019

Publication date PMC-release: 30 December 2019

Publication date Collection: 2019

Volume: 17

Electronic Location Identifier: 432

Affiliations

[1 ]GRID grid.21604.31, ISNI 0000 0004 0523 5263, Department of Transfusion Medicine, University Hospital of Salzburg (SALK), , Paracelsus Medical University, ; Salzburg, Austria

[2 ]GRID grid.21604.31, ISNI 0000 0004 0523 5263, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), , Paracelsus Medical University, ; Strubergasse 21, 5020 Salzburg, Austria

[3 ]GRID grid.21604.31, ISNI 0000 0004 0523 5263, Department of Laboratory Medicine, University Hospital of Salzburg (SALK), , Paracelsus Medical University, ; Salzburg, Austria

[4 ]GRID grid.21604.31, ISNI 0000 0004 0523 5263, Institute of Ecomedicine, , Paracelsus Medical University, ; Salzburg, Austria

[5 ]GRID grid.21604.31, ISNI 0000 0004 0523 5263, Cell Therapy Institute, , Paracelsus Medical University, ; Salzburg, Austria

Author information

Katharina Schallmoser http://orcid.org/0000-0002-7204-0058

Article

Publisher ID: 2183

DOI: 10.1186/s12967-019-02183-0

PMC ID: 6936143

PubMed ID: 31888679

SO-VID: 0211a082-25f0-4042-989c-f6aea64cd30d

License:

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 24 July 2019

Date accepted : 20 December 2019

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100010661, Horizon 2020 Framework Programme;

Award ID: 668724

Award ID: 731377

Award ID: 733006

Award Recipient : Dirk Strunk Katharina Schallmoser

Custom metadata

ScienceOpen disciplines: Medicine

Keywords: stromal cells,pooled human platelet lysate (phpl),fibrinogen,fetal bovine serum (fbs),mitotic bookmarking/transcription factors

Data availability:

ScienceOpen disciplines: Medicine

Keywords: stromal cells, pooled human platelet lysate (phpl), fibrinogen, fetal bovine serum (fbs), mitotic bookmarking/transcription factors

Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate

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Most cited references 57

A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells.

Single Cell RNA-Sequencing of Pluripotent States Unlocks Modular Transcriptional Variation

Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released.

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