Elsevier

Acta Biomaterialia

Volume 71, 15 April 2018, Pages 235-246
Acta Biomaterialia

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

https://doi.org/10.1016/j.actbio.2018.02.025Get rights and content

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.

Introduction

Heart valve disease is an increasing clinical burden associated with high morbidity and mortality [1], [2]. The prevalence of valvular disease is expected to triple by 2050 due to age-dependent degeneration of heart valves and rheumatic fever in developing countries [3], [4]. Congenital heart defects also contribute to the incidence of heart valve disease and occur in 1–2% of births [5].

With limited biological diagnostics or drugs to prevent heart valve disease, treatment is restricted to valve repair, valve replacement, or the Ross procedure. Valve replacement is often performed in older patients using either a mechanical or biological valve prosthesis [6]. While these implants improve survival and quality of life, the lack of cells is considered to be the main source of failure for mechanical or glutaraldehyde-fixed biological valves [7] and the main reason for their inability to grow and repair. Without the potential for somatic growth, pediatric patients are required to undergo several surgeries for valve refitting [8], [9]. Tissue engineered heart valves (TEHV) can potentially address the shortcomings of current implants by incorporating biomaterials with autologous cells to enable growth and biological integration [10].

Native heart valves are durable because valve cells maintain tissue homeostasis [11], and without these cells, mechanical or biological prostheses fail over time [7]. Within the valve there are two primary cell types: valve endothelial cells (VECs) and valve interstitial cells (VICs). The primary focus of this study will be on VICs, which are found throughout the three layers of the leaflet and are known to synthesize and remodel the extracellular matrix [11], [12], [13]. These cells are believed to mediate long-term valve durability but could play a role in heart valve disease progression [7]. Other cell types such as fibroblasts and smooth muscle cells are also believed to populate the leaflets and play a role in active matrix remodeling [14].

Despite recent advances in the field of TEHV, the challenge to identify a suitable cell source for seeding 3D scaffolds still remains [7], [14]. To ensure the success of the TEHV, various cell types have been investigated. These include xenogeneic, allogenic, and autologous cells. Examples of these cell types include bone marrow-derived cells, adipose-derived cells, and amniotic fluid cells from porcine, sheep, and human sources [15], [16], [17]. Xenogeneic and allogenic cells have been shown to evoke an immune response and may only be suitable for preclinical research [2], [18]. Meanwhile, autologous cells are the most appropriate cell source for seeding TEHV because they are patient-specific and depending on the cell source, they have the potential to be expanded and differentiated into other cell types.

More recently, mesenchymal stem cells (MSCs) derived from various sources such as bone marrow (BM) and adipocytes have been investigated as a potential cell source for TEHV [14], [19], [20]. This is because of their similar characteristics to smooth muscle cells and fibroblasts and their ability to differentiate into several cell types. While initial studies demonstrated promising results, a major limitation of MSCs derived from sources like bone marrow is a decrease in differentiation potential with expansion of in vitro cultures [21], [22], [23], which results in limited therapeutic efficacy.

An alternative cell source that maintains a higher level of stemness and can be readily expanded for clinical translation is induced pluripotent stem cells (iPSCs). Previous studies have shown that MSCs derived from human embryonic stem cells have the same cell surface phenotype compared to BM-derived MSCs [22]. Meanwhile, iPSC-derived MSCs (iMSCs) demonstrate trilineage differentiation [24]. With minimal senescence and higher telomerase activity potential [22], [25], iMSCs from iPSCs can be a potential cell source for TEHV. While only a few other groups have generated iMSCs from iPSCs, there is a need for safer transgene-free iPSCs and a feeder-free differentiation protocol for a more clinically translatable cell source.

While the outcomes of a TEHV depends on the cell source, the scaffold in which the cells are seeded can be utilized to direct cell phenotype and function. To recapitulate the ECM architecture in valve leaflets, a variety of natural and synthetic biomaterials have been explored [26]. An ideal scaffold for TEHV is one that provides the biological cues to promote cell migration, proliferation, differentiation, and spreading [27]. The scaffold should also allow for the exchange of oxygen and cellular waste, and stimulate ECM production and remodeling [28].

In particular, poly(ethylene glycol) (PEG) hydrogels can be designed to promote proper cell phenotype, proliferation, ECM production, and proteolytic degradation of the ECM. The goal is to generate a hydrogel that can enable cell adhesion and stimulate iMSCs to actively remodel the scaffold with ECM production [7]. Thus, the hydrogel network must accommodate the initial activation of iMSCs for active remodeling of the matrix, but over time, it must maintain cells in a quiescent fibroblast phenotype to prevent a pathological phenotype. Several groups have investigated PEG-diacrylate (PEGDA) as a hydrogel scaffold for TEHV applications [29], [30], [31], [32]. Thus, we investigated the maturation of iMSCs into VIC phenotype by encapsulating iMSCs into a 3D PEGDA hydrogel mimicking the microenvironment found in native valve leaflets.

The objective of this study is to develop a feeder-free protocol for differentiating iMSCs from integration-free iPSCs and to introduce these cells into a three-dimensional hydrogel to promote VIC phenotype, and ECM matrix production and remodeling. We hypothesize a feeder-free protocol for differentiating embryonic stem cells can be modified to differentiate iPSCs into iMSCs. The introduction of iMSCs into a 3D hydrogel commonly utilized to study VIC phenotype and ECM production will enable us to identify the potential of iMSCs as a cell source for TEHV.

Section snippets

Materials and methods

All media components were purchased from Thermo Fisher Scientific (Waltham, MA, US) unless otherwise stated.

iMSCs derived from iPSCs using a feeder-free approach demonstrate phenotypic similarity to human MSCs

To address the need for a suitable cell source for seeding TEHV, integration-free iPSCs were first generated from human dermal fibroblasts derived from a healthy child (Supplementary Fig. 1), then differentiated into iMSCs using a feeder-free protocol. The surface marker profile of iMSCs was characterized by the expressions of CD90, CD44, CD71, αSMA, and CD45 using flow cytometry. Fig. 1 shows that iMSCs have a 99.3%, 98.8%, 99.6%, and 95.9% positive expression of CD90, CD44, CD71, and αSMA,

Discussion

A major shortcoming for current valve prostheses is the absence of cells required for active repair and remodeling of the scaffold [7]. Valve cells play an important role in the tissue homeostasis, in particular VICs, which synthesize and remodel the extracellular matrix [11], [12], [13]. In the current study, we attempted to generate a patient-specific cell source for adequate seeding of TEHVs. Cells were generated using a feeder-free differentiation protocol that had been previously used for

Conclusion

Developing a suitable cell source for tissue engineering is a critical component for the success and durability of TEHV. In this study, we attempt to address this problem by differentiating iMSCs from iPSCs and then further maturing these cells into VIC-like cells by introducing these to a 3D culture designed to mimic the layers of the valve leaflets. First, a modified differentiation protocol was utilized to differentiate iPSCs into iMSCs using a feeder-free approach. Next, the PEGDA hydrogel

6. Acknowledgements

This work was supported by The Betkowski Family Research Fund, NSF Graduate Research Fellowship, American Heart Association Predoctoral Fellowship, NRSA NIH F31 Predoctoral Fellowship, Alfred P. Sloan Foundation, Goizueta Foundation to A.L.Y.N, and the Petit Scholar Program to S.L. The authors thank Jiahui Zhang for assisting with peptide conjugation and characterization. We also thank Joan Fernandez Esmerats for providing control cells. This research project was supported in part by the Emory

Disclosures

The authors have no conflicts to disclose.

References (53)

  • C.A. Durst et al.

    Flexural characterization of cell encapsulated PEGDA hydrogels with applications for tissue engineered heart valves

    Acta Biomater.

    (2011)
  • K.M. Mabry et al.

    Dynamic stiffening of poly(ethylene glycol)-based hydrogels to direct valvular interstitial cell phenotype in a three-dimensional environment

    Biomaterials

    (2015)
  • S. Ali et al.

    Micropatterning of poly(ethylene glycol) diacrylate hydrogels

    Methods Cell Biol.

    (2014)
  • S. Hinderer et al.

    Engineering of a bio-functionalized hybrid off-the-shelf heart valve

    Biomaterials

    (2014)
  • Y.Y. Zhou et al.

    Integration-free methods for generating induced pluripotent stem cells

    Genomics Proteomics Bioinf.

    (2013)
  • A. Hasan et al.

    Biomechanical properties of native and tissue engineered heart valve constructs

    J. Biomech.

    (2014)
  • F. Yang et al.

    The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells

    Biomaterials

    (2005)
  • A.C. Liu et al.

    The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology

    Am. J. Pathol.

    (2007)
  • P.M. Taylor et al.

    The cardiac valve interstitial cell

    Int. J. Biochem. Cell Biol.

    (2003)
  • F. Colazzo et al.

    Extracellular matrix production by adipose-derived stem cells: implications for heart valve tissue engineering

    Biomaterials

    (2011)
  • E.J. Benjamin et al.

    Heart disease and stroke statistics-2017 update: a report from the American Heart Association

    Circulation

    (2017)
  • J.J. Takkenberg et al.

    The need for a global perspective on heart valve disease epidemiology. The SHVD working group on epidemiology of heart valve disease founding statement

    J. Heart Valve Dis.

    (2008)
  • J. Hoffman

    The global burden of congenital heart disease

    Cardiovasc. J. Afr.

    (2013)
  • M.S. Sacks et al.

    Bioengineering challenges for heart valve tissue engineering

    Annu. Rev. Biomed. Eng.

    (2009)
  • K. Mendelson et al.

    Heart valve tissue engineering: concepts, approaches, progress, and challenges

    Ann. Biomed. Eng.

    (2006)
  • A.H. Chester et al.

    The living aortic valve: From molecules to function

    Glob. Cardiol. Sci. Pract.

    (2014)
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