2. Academic Validation
  3. Dynamic three dimensional environment for efficient and large scale generation of smooth muscle cells from hiPSCs

Dynamic three dimensional environment for efficient and large scale generation of smooth muscle cells from hiPSCs

  • Stem Cell Res Ther. 2024 Dec 3;15(1):463. doi: 10.1186/s13287-024-04053-z.
Akazha Green 1,  Yuhua Wei 1,  Jason M Warram 1 2,  Yolanda E Hartman 1 2,  Xiaoxiao Geng 1,  Thanh Nguyen 1,  Lei Ye 3,  Jianyi Zhang 4 5
Affiliations

Affiliations

  • 1 Department of Biomedical Engineering, The University of Alabama at Birmingham, Volker Hall, 1670 University Boulevard, Birmingham, AL, 35255, USA.
  • 2 Department of Otolaryngology, University of Alabama at Birmingham, AL., Birmingham, 35255, USA.
  • 3 Department of Biomedical Engineering, The University of Alabama at Birmingham, Volker Hall, 1670 University Boulevard, Birmingham, AL, 35255, USA. lye@uab.edu.
  • 4 Department of Biomedical Engineering, The University of Alabama at Birmingham, Volker Hall, 1670 University Boulevard, Birmingham, AL, 35255, USA. jayzhang@uab.edu.
  • 5 Department of Medicine, Division of Cardiovascular Disease, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35255, USA. jayzhang@uab.edu.
PMID: 39627821 DOI: 10.1186/s13287-024-04053-z
Abstract

Background: Chronic ischemic limb disease often leads to amputation, which remains a significant clinical problem. Smooth-muscle cells (SMCs) are crucially involved in the development and progression of many cardiovascular diseases, but studies with primary human SMCs have been limited by a lack of availability. Here, we evaluated the efficiency of two novel protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into SMCs and assessed their potency for the treatment of ischemic limb disease.

Methods: hiPSCs were differentiated into SMCs via a conventional two-dimensional (2D) protocol that was conducted entirely with cell monolayers, or via two protocols that consisted of an initial five-day three-dimensional (3D) spheroid phase followed by a six-day 2D monolayer phase (3D + 2D differentiation). The 3D phases were conducted in shaker flasks on an orbital shaker (the 3D + 2D shaker protocol) or in a PBS bioreactor (the 3D + 2D bioreactor protocol). Differentiation efficiency was evaluated via the expression of SMC markers (smooth-muscle actin [SMA], smooth muscle protein 22 [SM22], and Calponin-1), and the biological activity of the differentiated hiPSC-SMCs was evaluated via in-vitro assessments of migration (scratch assay), contraction in response to the treatment with a prostaglandin H2 analog (U46619), and tube formation on Geltrex, as well as in-vivo measurements of perfusion (fluorescence angiography) and vessel density in the limbs of mice that were treated with hiPSC-SMCs after experimentally induced hind-limb ischemia (HLI).

Results: Both 3D + 2D protocols yielded > 5.6 × 107 hiPSC-SMCs/differentiation, which was ~ nine-fold more than that produced via 2D differentiation, and flow cytometry analyses confirmed that > 98% of the 3D + 2D-differentiated hiPSC-SMCs expressed SMA, > 81% expressed SM22, and > 89% expressed Calponin-1. hiPSC-SMCs obtained via the 3D + 2D shaker protocol also displayed typical SMC-like migratory, contraction, and tube-formation activity in-vitro and significantly improved measurements of perfusion, vessel density, and SMA-positive arterial density in the ischemic limb of mouse HLI model.

Conclusions: Our dynamic 3D + 2D protocols produced an exceptionally high yield of hiPSC-SMCs. Transplantation of these hiPSC-SMCs results in significantly improved recovery of ischemic limb after ischemic injury in mice.

Keywords

Cell differentiation; Human induced pluripotent stem cells; Ischemic limb disease; Smooth muscle cells.

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