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

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Sandra Laner-Plamberger ◽  
Michaela Oeller ◽  
Cornelia Mrazek ◽  
Arnulf Hartl ◽  
Alina Sonderegger ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Stromal Cells ◽  
Biochemical Parameters ◽  
Stromal Cell ◽  
Human Platelet ◽  
Culture Conditions ◽  
Platelet Lysate ◽  
In Vitro Differentiation ◽  
Human Platelet Lysate

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 Fe3+ 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.

scholarly journals Human Platelet Lysate Maintains Stemness of Umbilical Cord-Derived Mesenchymal Stromal Cells and Promote Lung Repair in Rat Bronchopulmonary Dysplasia

2021 ◽  
Vol 9 ◽  
Author(s):  
Guilian Liao ◽  
Yan Liao ◽  
Duanduan Li ◽  
Zeqin Fu ◽  
Shiduo Wu ◽  
...  
Keyword(s):  
Umbilical Cord ◽  
Bronchopulmonary Dysplasia ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Paracrine Factors ◽  
Lung Repair ◽  
Human Platelet Lysate

Mesenchymal stromal cells (MSCs) show potential for treating preclinical models of newborn bronchopulmonary dysplasia (BPD), but studies of their therapeutic effectiveness have had mixed results, in part due to the use of different media supplements for MSCs expansion in vitro. The current study sought to identify an optimal culture supplement of umbilical cord-derived MSCs (UC-MSCs) for BPD therapy. In this study, we found that UC-MSCs cultured with human platelet lysate (hPL-UCMSCs) were maintained a small size from Passage 1 (P1) to P10, while UC-MSCs cultured with fetal bovine serum (FBS-UCMSCs) became wide and flat. Furthermore, hPL was associated with lower levels of senescence in UC-MSCs during in vitro expansion compared with FBS, as indicated by the results of β-galactosidase staining and measures of senescence-related genes (CDKN2A, CDKN1A, and mTOR). In addition, hPL enhanced the proliferation and cell viability of the UC-MSCs and reduced their doubling time in vitro. Compared with FBS-UCMSCs, hPL-UCMSCs have a greater potential to differentiate into osteocytes and chondrocytes. Moreover, using hPL resulted in greater expression of Nestin and specific paracrine factors (VEGF, TGF-β1, FGF2, IL-8, and IL-6) in UC-MSCs compared to using FBS. Critically, we also found that hPL-UCMSCs are more effective than FBS-UCMSCs for the treatment of BPD in a rat model, with hPL leading to improvements in survival rate, lung architecture and fibrosis, and lung capillary density. Finally, qPCR of rat lung mRNA demonstrated that hPL-UCMSCs had lower expression levels of inflammatory factors (TNF-α and IL-1β) and a key chemokine (MCP-1) at postnatal day 10, and there was significant reduction of CD68+ macrophages in lung tissue after hPL-UCMSCs transplantation. Altogether, our findings suggest that hPL is an optimal culture supplement for UC-MSCs expansion in vitro, and that hPL-UCMSCs promote lung repair in rat BPD disease.

scholarly journals Stromal cells in myeloid and lymphoid long-term bone marrow cultures can support multiple hemopoietic lineages and modulate their production of hemopoietic growth factors

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Culture Conditions ◽  
B Lymphopoiesis ◽  
Bone Marrow Cultures ◽  
Factor Secretion ◽  
Marrow Cultures

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.

scholarly journals A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro

Reproduction ◽  
2011 ◽  
Vol 141 (6) ◽  
pp. 809-820 ◽  
Author(s):  
Candace M Tingen ◽  
Sarah E Kiesewetter ◽  
Jennifer Jozefik ◽  
Cristina Thomas ◽  
David Tagler ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture System ◽  
Stromal Cell ◽  
Ovarian Follicle ◽  
3D Culture ◽  
Culture Conditions ◽  
Growth And Survival ◽  
3D Culture System

Innovations in in vitro ovarian follicle culture have revolutionized the field of fertility preservation, but the successful culturing of isolated primary and small secondary follicles remains difficult. Herein, we describe a revised 3D culture system that uses a feeder layer of ovarian stromal cells to support early follicle development. This culture system allows significantly improved primary and early secondary follicle growth and survival. The stromal cells, consisting mostly of thecal cells and ovarian macrophages, recapitulate the in vivo conditions of these small follicles and increase the production of androgens and cytokines missing from stromal cell-free culture conditions. These results demonstrate that small follicles have a stage-specific reliance on the ovarian environment, and that growth and survival can be improved in vitro through a milieu created by pre-pubertal ovarian stromal cell co-culture.

Expansion of Adipose Mesenchymal Stromal Cells is Affected by Human Platelet Lysate and Plating Density

2011 ◽  
Vol 20 (9) ◽  
pp. 1409-1422 ◽  
Author(s):  
Dominik Cholewa ◽  
Thomas Stiehl ◽  
Anne Schellenberg ◽  
Gudrun Bokermann ◽  
Sylvia Joussen ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Human Platelet ◽  
Platelet Lysate ◽  
Plating Density ◽  
Human Platelet Lysate
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