Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 15:71:235-246.
doi: 10.1016/j.actbio.2018.02.025. Epub 2018 Mar 2.

Human iPSC-derived mesenchymal stem cells encapsulated in PEGDA hydrogels mature into valve interstitial-like cells

Aline L Y Nachlas  1 , Siyi Li  1 , Rajneesh Jha  2 , Monalisa Singh  2 , Chunhui Xu  3 , Michael E Davis  4
Affiliations

Human iPSC-derived mesenchymal stem cells encapsulated in PEGDA hydrogels mature into valve interstitial-like cells

Aline L Y Nachlas et al. Acta Biomater. .

Abstract

Despite recent advances in tissue engineered heart valves (TEHV), a major challenge is identifying a cell source for seeding TEHV scaffolds. Native heart valves are durable because valve interstitial cells (VICs) maintain tissue homeostasis by synthesizing and remodeling the extracellular matrix. This study demonstrates that induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (iMSCs) can be derived from iPSCs using a feeder-free protocol and then further matured into VICs by encapsulation within 3D hydrogels. The differentiation efficiency was characterized using flow cytometry, immunohistochemistry staining, and trilineage differentiation. Using our feeder-free differentiation protocol, iMSCs were differentiated from iPSCs and had CD90+, CD44+, CD71+, αSMA+, and CD45- expression. Furthermore, iMSCs underwent trilineage differentiation when cultured in induction media for 21 days. iMSCs were then encapsulated in poly(ethylene glycol)diacrylate (PEGDA) hydrogels grafted with adhesion peptide (RGDS) to promote remodeling and further maturation into VIC-like cells. VIC phenotype was assessed by the expression of alpha-smooth muscle actin (αSMA), vimentin, and collagen production after 28 days. When MSC-derived cells were encapsulated in PEGDA hydrogels that mimic the leaflet modulus, a decrease in αSMA expression and increase in vimentin was observed. In addition, iMSCs synthesized collagen type I after 28 days in 3D hydrogel culture. Thus, the results from this study suggest that iMSCs may be a promising cell source for TEHV.

Statement of significance: Developing a suitable cell source is a critical component for the success and durability of tissue engineered heart valves. The significance of this study is the generation of iPSCs-derived mesenchymal stem cells (iMSCs) that have the capacity to mature into valve interstitial-like cells when introduced into a 3D cell culture designed to mimic the layers of the valve leaflet. iMSCs were generated using a feeder-free protocol, which is one major advantage over other methods, as it is more clinically relevant. In addition to generating a potential new cell source for heart valve tissue engineering, this study also highlights the importance of a 3D culture environment to influence cell phenotype and function.

Keywords: Hydrogel; Induced pluripotent stem cells; Mesenchymal stem cells; PEG; Tissue engineering heart valves.

PubMed Disclaimer

Conflict of interest statement

Disclosures

The authors have no conflicts to disclose.

Figures

Figure 1
Figure 1. Characterization of iMSCs differentiation efficiency via flow cytometry
A) iMSCs were stained for positive expression of CD90, CD44, CD71, and αSMA surface markers. No expression of CD45 was observed. Blue peaks = stained cells. Black peaks = unstained cells. B) The expression of each marker was quantified by gating population of iMSCs and comparing to non-stained cells. N = 3–4.
Figure 2
Figure 2. Trilineage Differentiation of iMSCs
A) iMSCs were cultured in adipogenic, osteogenic, chondrogenic induction media for 21 days. Control iMSCs were cultured in MSC media. iMSCs demonstrate the ability to differentiate into adipocytes by evidence of intracellular fat deposits stained by Oil Red O. iMSCs cultured in osteogenic media led to high levels of extracellular calcium deposition stained in bright red using Alizarin Red S. High levels of toluidine blue staining in observed in iMSCs treated with chondrogenic media. B) The absorbency of each stain was quantified. Scale bar 50 μm. *p<0.05; **p<0.01; ***p<0.001; t-test.
Figure 3
Figure 3. Characterization of iMSC phenotype
iMSCs and human MSCs (control) were immunostained with DAPI, F-Actin, and αSMA after 24 hours. iMSCs had comparable expression of αSMA and F-actin stress fibers to human MSCs. Scale bar 50 μm.
Figure 4
Figure 4. Mechanical properties of PEGDA hydrogels
Compression testing was used to determine the bulk modulus of 5% v/w PEGDA hydrogels with 5mM RGDS, crosslinked with white-light for 1-minute. The elastic modulus in PEGDA only hydrogels was an average of 5.6 kPa. While cell-laden PEGDA hydrogels had a modulus of 30 kPa, which decreased to 3.7 kPa after 28 days. ****p<0.0001; N = 8 –10.
Figure 5
Figure 5. Cell viability of iMSCs encapsulated in 5% PEGDA hydrogels
iMSCs were encapsulated into PEGDA hydrogels 5% w/v with 5 mM RGDS. Hydrogels were cultured for 1 or 7 days. Cell viability was 93% after 1 day of encapsulation and maintained at 77% after 7 days. There was no statistical significance between day 1 or day 7. Green is Calcein AM. Red represents ethidium homodimer. N = 3. Scale bar 50 μm.
Figure 6
Figure 6. iMSC expression of αSMA and vimentin after 28 days
iMSCs, VICs, and HDFs were encapsulated into PEGDA hydrogels 5% w/v with 5 mM RGDS for 28 days. Fluorescence for αSMA and vimentin was quantified for each cell type at day 1 and day 28. Student t-test was conducted to comparing day 1 to day 28. DAPI=Blue; Vimentin= Green; αSMA = Red. Scale bar 50 μm. **p<0.01; ***p<0.001 t-test; N = 4–8.
Figure 7
Figure 7. iMSC expression of periostin and calponin after 28 days
All three cell types, iMSCs, VICs, and HDFs were encapsulated into 5% w/v PEGDA hydrogels with 5 mM RGDS for 28 days. Fluorescence for periostin and calponin was quantified for each cell type at day 1 and day 28. Student t-test was conducted to compare changes in expression over time. DAPI=Blue; Periostin= Green; Calponin = Red. Scale bar 50 μm. **p<0.01 t-test; N = 4–8.
Figure 8
Figure 8. iMSC Collagen Type I Deposition in PEGDA Hydrogels
iMSCs, VICs, and HDFs were encapsulated into PEGDA hydrogels 5% w/v with 5 mM RGDS. Hydrogels were cultured for 1 and 28 days before being stained for collagen type I. Over time, iMSCs begin to deposit collagen. Student t-test was conducted to compare changes in expression over time. DAPI = Blue. Cell membrane = Green. Collagen type I= Red. ****p<0.0001 t-test; N = 3. Scale bar 50 μm.
Figure 9
Figure 9. Quantification of DNA content and collagen in cell-laden PEGDA hydrogels
iMSCs were encapsulated into 5% w/v PEGDA hydrogels with 5 mM RGDS for 1, 7, 14, and 28 days. A) Using the PicoGreen assay, the DNA content remained unchanged at all time points. N = 4. B) Collagen content was determined by hydroxyproline assay and normalized by wet weight of hydrogel. *p<0.05 vs day 1; ANOVA followed by Dunnett’s post-test. N = 4–5.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, de Ferranti SD, Floyd J, Fornage M, Gillespie C, Isasi CR, Jimenez MC, Jordan LC, Judd SE, Lackland D, Lichtman JH, Lisabeth L, Liu S, Longenecker CT, Mackey RH, Matsushita K, Mozaffarian D, Mussolino ME, Nasir K, Neumar RW, Palaniappan L, Pandey DK, Thiagarajan RR, Reeves MJ, Ritchey M, Rodriguez CJ, Roth GA, Rosamond WD, Sasson C, Towfighi A, Tsao CW, Turner MB, Virani SS, Voeks JH, Willey JZ, Wilkins JT, Wu JH, Alger HM, Wong SS, Muntner P, C. American Heart Association Statistics, S. Stroke Statistics Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation. 2017;135(10):e146–e603. - PMC - PubMed
    1. Jana S, Lerman A. Bioprinting a cardiac valve. Biotechnol Adv. 2015;33(8):1503–21. - PubMed
    1. Takkenberg JJ, Rajamannan NM, Rosenhek R, Kumar AS, Carapetis JR, Yacoub MH, D. Society for Heart Valve The need for a global perspective on heart valve disease epidemiology. The SHVD working group on epidemiology of heart valve disease founding statement. The Journal of heart valve disease. 2008;17(1):135–9. - PubMed
    1. Marijon E, Mirabel M, Celermajer DS, Jouven X. Rheumatic heart disease. Lancet. 2012;379(9819):953–64. - PubMed
    1. Hoffman J. The global burden of congenital heart disease. Cardiovascular journal of Africa. 2013;24(4):141–5. - PMC - PubMed

Publication types

MeSH terms

Substances