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
  • 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. [email protected].
  • 4. Department of Biomedical Engineering, The University of Alabama at Birmingham, Volker Hall, 1670 University Boulevard, Birmingham, AL, 35255, USA. [email protected].
  • 5. Department of Medicine, Division of Cardiovascular Disease, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35255, USA. [email protected].
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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