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Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

Naaijkens BA, Niessen HW, Prins HJ, Krijnen PA, Kokhuis TJ, de Jong N, van Hinsbergh VW, Kamp O, Helder MN, Musters RJ, van Dijk A, Juffermans LJ - Cell Tissue Res. (2012)

Bottom Line: We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC.PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay.In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. b.naaijkens@vumc.nl

ABSTRACT
Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

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Presence of markers indicative for cardiomyocyte differentiation. a–l Microscopical images of staining of non-stimulated (a, b) and 5-aza-2-deoxycytidin stimulated ASC, both FBS- and PL-cultured on cytospin slides for desmin (c, d), troponin T(e, f), α-actinin (g, h), MLC-2α (i, j) and connexin43(k, l) at day 10. m–p Microscopical images of Cx43 staining of paraffin-embedded human adult tissue. Cx43 is present in cardiomyocytes (p, arrows), not in skeletal muscle (o). PBS controls were also negative (a, b). q–t Microscopical images of Cx43 staining of ASC on chamber slides. Cx43 is present in 5-aza-2-deoxycytidin stimulated ASC at day 10, both FBS- (s) and PL-cultured (t), but not in non-differentiated control ASC (q–r)
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Fig5: Presence of markers indicative for cardiomyocyte differentiation. a–l Microscopical images of staining of non-stimulated (a, b) and 5-aza-2-deoxycytidin stimulated ASC, both FBS- and PL-cultured on cytospin slides for desmin (c, d), troponin T(e, f), α-actinin (g, h), MLC-2α (i, j) and connexin43(k, l) at day 10. m–p Microscopical images of Cx43 staining of paraffin-embedded human adult tissue. Cx43 is present in cardiomyocytes (p, arrows), not in skeletal muscle (o). PBS controls were also negative (a, b). q–t Microscopical images of Cx43 staining of ASC on chamber slides. Cx43 is present in 5-aza-2-deoxycytidin stimulated ASC at day 10, both FBS- (s) and PL-cultured (t), but not in non-differentiated control ASC (q–r)

Mentions: Since PL-cultured ASC differed in several assays from FBS-cultured ASC, we studied whether they were still capable of differentiating towards cardiomyocytes. To determine this, ASC were stimulated with 5-aza-2-deoxycytidin. After 10 and 21 days, the occurrence of different markers present on cardiomyocytes and the microscopical morphology were analyzed. First, we showed that desmin, α-actinin, troponin T, myosin light chain-2α (MLC-2α), and Connexin43 (Cx43) were present in stimulated FBS- and PL-cultured ASC, and not in non-stimulated ASC (Fig. 5a–l). Desmin, α-actinin, troponin T, and MLC-2α are also present on skeletal muscle; however, we demonstrated that Cx43 was only present in human adult cardiomyocytes, and absent in human adult skeletal muscle (Fig. 5m–p). In addition, we found that stimulation also induced morphological changes shown in stimulated ASC in chamberslides that were stained with Cx43: non-stimulated ASC displayed fibroblast morphology, whereas the stimulated ASC, both FBS- and PL-cultured, appeared much larger and more elongated resembling muscle cells (Fig. 5q–t). Furthermore, we quantified the number of positive cells for all markers (Fig. 6a–e). Between 65 and 86% of both FBS- and PL-cultured ASC stained positive for all markers, which was significantly higher compared with non-stimulated ASC (1–14%; p < 0.05). No significant differences were found between 10 and 21 days after stimulation, nor between FBS- and PL-cultured ASC, indicating no difference in differentiation capacity (Fig. 6). Notably, no dedifferentiation was found after 12 weeks for either FBS- or PL-cultured ASC (data not shown).Fig. 5


Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications.

Naaijkens BA, Niessen HW, Prins HJ, Krijnen PA, Kokhuis TJ, de Jong N, van Hinsbergh VW, Kamp O, Helder MN, Musters RJ, van Dijk A, Juffermans LJ - Cell Tissue Res. (2012)

Presence of markers indicative for cardiomyocyte differentiation. a–l Microscopical images of staining of non-stimulated (a, b) and 5-aza-2-deoxycytidin stimulated ASC, both FBS- and PL-cultured on cytospin slides for desmin (c, d), troponin T(e, f), α-actinin (g, h), MLC-2α (i, j) and connexin43(k, l) at day 10. m–p Microscopical images of Cx43 staining of paraffin-embedded human adult tissue. Cx43 is present in cardiomyocytes (p, arrows), not in skeletal muscle (o). PBS controls were also negative (a, b). q–t Microscopical images of Cx43 staining of ASC on chamber slides. Cx43 is present in 5-aza-2-deoxycytidin stimulated ASC at day 10, both FBS- (s) and PL-cultured (t), but not in non-differentiated control ASC (q–r)
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Fig5: Presence of markers indicative for cardiomyocyte differentiation. a–l Microscopical images of staining of non-stimulated (a, b) and 5-aza-2-deoxycytidin stimulated ASC, both FBS- and PL-cultured on cytospin slides for desmin (c, d), troponin T(e, f), α-actinin (g, h), MLC-2α (i, j) and connexin43(k, l) at day 10. m–p Microscopical images of Cx43 staining of paraffin-embedded human adult tissue. Cx43 is present in cardiomyocytes (p, arrows), not in skeletal muscle (o). PBS controls were also negative (a, b). q–t Microscopical images of Cx43 staining of ASC on chamber slides. Cx43 is present in 5-aza-2-deoxycytidin stimulated ASC at day 10, both FBS- (s) and PL-cultured (t), but not in non-differentiated control ASC (q–r)
Mentions: Since PL-cultured ASC differed in several assays from FBS-cultured ASC, we studied whether they were still capable of differentiating towards cardiomyocytes. To determine this, ASC were stimulated with 5-aza-2-deoxycytidin. After 10 and 21 days, the occurrence of different markers present on cardiomyocytes and the microscopical morphology were analyzed. First, we showed that desmin, α-actinin, troponin T, myosin light chain-2α (MLC-2α), and Connexin43 (Cx43) were present in stimulated FBS- and PL-cultured ASC, and not in non-stimulated ASC (Fig. 5a–l). Desmin, α-actinin, troponin T, and MLC-2α are also present on skeletal muscle; however, we demonstrated that Cx43 was only present in human adult cardiomyocytes, and absent in human adult skeletal muscle (Fig. 5m–p). In addition, we found that stimulation also induced morphological changes shown in stimulated ASC in chamberslides that were stained with Cx43: non-stimulated ASC displayed fibroblast morphology, whereas the stimulated ASC, both FBS- and PL-cultured, appeared much larger and more elongated resembling muscle cells (Fig. 5q–t). Furthermore, we quantified the number of positive cells for all markers (Fig. 6a–e). Between 65 and 86% of both FBS- and PL-cultured ASC stained positive for all markers, which was significantly higher compared with non-stimulated ASC (1–14%; p < 0.05). No significant differences were found between 10 and 21 days after stimulation, nor between FBS- and PL-cultured ASC, indicating no difference in differentiation capacity (Fig. 6). Notably, no dedifferentiation was found after 12 weeks for either FBS- or PL-cultured ASC (data not shown).Fig. 5

Bottom Line: We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC.PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay.In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. b.naaijkens@vumc.nl

ABSTRACT
Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically, transmission of pathogens and antibody development against FBS are possible. In this study, we investigated whether FBS can be substituted by human platelet lysate (PL) in ASC culture, without affecting functional capacities particularly important for cardiac repair application of ASC. We found that PL-cultured ASC had a significant 3-fold increased proliferation rate and a significantly higher attachment to tissue culture plastic as well as to endothelial cells compared with FBS-cultured ASC. PL-cultured ASC remained a significant 25% smaller than FBS-cultured ASC. Both showed a comparable surface marker profile, with the exception of significantly higher levels of CD73, CD90, and CD166 on PL-cultured ASC. PL-cultured ASC showed a significantly higher migration rate compared with FBS-cultured ASC in a transwell assay. Finally, FBS- and PL-cultured ASC had a similar high capacity to differentiate towards cardiomyocytes. In conclusion, this study showed that culturing ASC is more favorable in PL-supplemented medium compared with FBS-supplemented medium.

Show MeSH
Related in: MedlinePlus