Int J Stem Cells.  2020 Mar;13(1):151-162. 10.15283/ijsc19004.

Hyaluronan Induces a Mitochondrial Functional Switch in Fast-Proliferating Human Mesenchymal Stem Cells

• Solis MA
• Wei YH
• Chang CH
• Yu CH
• Huang LL

• KMID: 2500574
• DOI: http://doi.org/10.15283/ijsc19004

Abstract

Background and Objectives
Hyaluronan preserves the proliferation and differentiation potential of mesenchymal stem cells. Supplementation of low-concentration hyaluronan (SHA) in stem cells culture medium increases its proliferative rate, whereas coated-surface hyaluronan (CHA) maintains cells in a slow-proliferating mode. We have previously demonstrated that in CHA, the metabolic proliferative state of stem cells was influenced by upregulating mitochondrial biogenesis and function. However, the effect of SHA on stem cells’ energetic status remains unknown. In this study, we demonstrate the effect that low-concentration SHA at 0.001 mg/ml (SHA0.001) and high-concentration SHA at 5 mg/ml (SHA5) exert on stem cells’ mitochondrial function compared with CHA and noncoated tissue culture surface (control).
Methods and Results
Fast-proliferating human placenta-derived mesenchymal stem cells (PDMSCs) cultured on SHA0.001 exhibited reduced mitochondrial mass, lower mitochondrial DNA copy number, and lower oxygen consumption rate compared with slow-proliferating PDMSCs cultured on CHA at 5.0 (CHA5) or 30 μg/cm2 (CHA30). The reduced mitochondrial biogenesis observed in SHA0.001 was accompanied by a 2-fold increased ATP content and lactate production, suggesting that hyaluronan-induced fast-proliferating PDMSCs may rely less on mitochondrial function as an energy source and induce a mitochondrial functional switch to glycolysis.
Conclusions
PDMSCs cultured on both CHA and SHA exhibited a reduction in reactive oxygen species levels. The results from this study clarify our understandings on the effect of hyaluronan on stem cells and provide important insights into the effect of distinct supplementation methods used during cell therapies.

Keyword

Hyaluronan; Mesenchymal stem cells; Mitochondria; Cellular proliferation

Figure

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  Fig. 1 PDMSCs possess plastic adherence characteristics, differentiate into MSC specific lineages, and express MSC CD Markers. PDMSCs cultured on control surface contained (A) fibroblastic morphology and plastic adherence characteristic; were assayed for differentiation ability by 32-day culture in (B) adipogenesis, (C) chondrogenesis, and (D) osteogenesis differentiation medium; and (E) positively express the MSC CD markers CD73, CD90, and CD105. Images obtained after oil red, alcian blue, and alizarin red, respectively. PE: Phycoerythrin, FITC: Fluorescein Isothiocyanate, APC: Allophycocyanin. Scale bar: 100 μm.

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  Fig. 2 Different hyaluronan substratum exerted changes in PDMSCs cellular proliferation and morphology. (A) Cumulative population doubling of PDMSCs cultured for 34 days on control, medium supplemented hyaluronan (SHA 0.001, 0.01, 1, and 5 mg/ml), or coated hyaluronan (CHA 0.5, 3, 5, 30 μg/cm2). The drawing represents the cumulative population doublings with time where each dot represents one passage approximately every 4 days. Cumulative population doubling is expressed as mean±SD of at least triplicates; (B) cell growth curve and (C) morphological changes of PDMSCs cultured for 5 days in control, SHA 0.001 and 5 mg/ml; or CHA 5 and 30 μg/cm2. Scale bar: 100 μm. SHA: medium supplemented hyaluronan, CHA: coated hyaluronan. Three independent experiments were performed in control and experimental groups and data are represented in mean±SD.

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  Fig. 3 Different hyaluronan substratum exerted changes in mitochondrial distribution of PDMSCs. Successful clones for lentivector pAS3W-DsRed2-mito after ligation of the vectors pLKOAS3w.puro and pDsRed2-mito. (A) PCR gels showed successful clone band at 800 bp; and sequence alignment of 99% from restriction enzyme cutting sites of NheI at 96 bp and PmeI at 892 bp. (B) Transfection of PDMSCs with pAS3W-DsRed2-mito enabled red fluorescence labelling of mitochondria specifically. Lentivector containing DsRed-mito showed fluorescence punctuated patterns when transfected into PDMSCs, whereas lentivector with only DsRed showed a diffused fluorescence pattern; (C) mitochondrial localization of PDMSCs cultured for 5 days on control surfaces, SHA 0.001 and SHA 5 mg/ml or CHA 5 and 30 μg/cm2. (D) Confocal images of mitochondrial distribution in hyaluronan-supplemented PDMSCs. Red fluorescence represents mitochondria through PDMSCs transfected with expression plasmid pAS3W-DsRed2-mito; blue fluorescence represents nucleus through DAPI staining. Scale bar: 100 μm. SHA: medium supplemented hyaluronan, CHA: coated hyaluronan, DAPI: 4’,6-diamidino-2-phenylindole.

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  Fig. 4 Hyaluronan-induced fast-proliferative PDMSC had lower mitochondrial biogenesis. (A) Mitochondrial mass; (B) mtDNA copy number; All data is expressed as mean±SD of at least three replicates and analyzed by the paired t-test (*p<0.05, **p<0.01). Mitochondrial mass was analyzed by using PDMSCs transfected with expression plasmid pAS3w-DsRed2-mito. SHA: medium supplemented hyaluronan, CHA: coated hyaluronan, mtDNA: mitochondrial DNA, RFU: relative fluorescence unit.

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  Fig. 5 Higher ATP content and lactate production in hyaluronan-induced fast-proliferative PDMSCs. (A) ATP content; (B) OCR; (C) lactate production; (D) LDHA levels; and (E) ROS levels of PDMSCs cultured on control surface, SHA (0.001 mg/ml and 5 mg/ml), or CHA (5 and 30 μg/cm2). All data is expressed as mean±SD of at least three replicates and analyzed by paired t-test (*p<0.05, **p<0.01). SHA: medium supplemented hyaluronan, CHA: coated hyaluronan, ATP: adenosine triphosphate, OCR: oxygen consumption rate, ROS: reactive oxygen species, RFU: relative fluorescence unit, H2O2: hydrogen peroxide, O2−: superoxide anion.

Reference

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