Sulfated Alginate Reduces Pericapsular Fibrotic Overgrowth on Encapsulated cGMP-Compliant hPSC-Hepatocytes in Mice

Abstract

Intra-peritoneal placement of alginate encapsulated human induced pluripotent stem cell-derived hepatocytes (hPSC-Heps) represents a potential new bridging therapy for acute liver failure. One of the rate-limiting steps that needs to be overcome to make such a procedure more efficacious and safer is to reduce the accumulation of fibrotic tissue around the encapsulated cells to allow the free passage of relevant molecules in and out for metabolism. Novel chemical compositions of alginate afford the possibility of achieving this aim. We accordingly used sulfated alginate and demonstrated that this material reduced fibrotic overgrowth whilst not impeding the process of encapsulation nor cell function. Cumulatively, this suggests sulfated alginate could be a more suitable material to encapsulate hPSC-hepatocyte prior to human use.

Keywords: PFO; acute liver failure; cGMP; hPSC; immunogenicity; immunoisolation; pluripotent stem cell-derived hepatocytes; sulfated alginate.

FIGURE 1

Generation of sulfated alginate and alginate microbeads. (A) Sulfation of the alginate by treatment with chlorosulfonic acid in formamide (B) Encapsulation of PSC-Hep spheroids. The spheroids in a homogenous mixture of calcium-free alginate solution are pumped out of the encapsulator nozzle. The electrostatic accelerator ring pulls the droplets downwards resulting in a smaller droplet diameter. Subsequently, the cell-alginate droplets are collected in bivalent cation (Ca²⁺ and Ba²⁺; shown as 2+)-containing solution which promotes crosslinking of predominantly the blocks of L-guluronic acid in the alginate, and generation of solid-phase alginate beads. The alginate matrix allows diffusion of oxygen, nutrients and secreted cell products, and provides a barrier against infiltration of immune cells. (C) Gross morphology of alginate microbeads by brightfield microscopy. Unmodified UP-LVG alginate microbeads (left), 40% sulfated alginate microbeads (middle) and hPSC-Heps spheroids encapsulated in 40% sulfated alginate (right) all showed regular morphology. Scale bars: 500 μm.

FIGURE 2

Characterization of sulfated alginate microbeads. (A) Size distribution of encapsulated hPSC-Hep spheroids. The boxplots and histograms represent the diameter measurements of unmodified UP-LVG (gray; n = 34) and sulfated alginate (orange; n = 34). The dashed lines represent the median microbead diameter for each alginate type. (B) Permeability of the unmodified UP-LVG and sulfated alginate microbeads (n = 11). Selection of representative CLSM images showing different degrees of permeation of FITC-dextran probes at different molecular weights (top). Quantification of FITC-dextran permeation normalized to 4 kDa (100% permeation) and 2 MDa (0% permeation) (bottom). Scale bars: 500 μm. (*p< 0.05).

FIGURE 3

The functionality of encapsulated hPSC-Hep spheroids. (A) Comparison of the effects of non-modified and modified alginate microbeads on albumin secretion of encapsulated hPSC-Hep spheroids (n = 5). (B) Comparison of ureagenesis of hPSC-Hep spheroids encapsulated in UP LVG alginate and sulfated alginate (n = 3). (p**<0.01, ns = no significant difference).

FIGURE 4

Ex vivo assessment of pericapsular fibrotic outgrowth on the non-modified and sulfated alginate microbeads. (A) Brightfield microscopy images of alginate microbeads retrieved from mice 10 days after intraperitoneal implantation. hPSC-Heps spheroids encapsulated in sulfated alginate show a lower degree of PFO compared to the spheroids encapsulated in non-modified alginate. Similarly, sulfated alginate empty microbeads show a lower degree of PFO than empty non-modified alginate. Scale bars: 500 μm (B) Stack bars showing the average degree of PFO for the alginate microbeads retrieved from the peritoneal cavity of mice at 10 days and 20 days post-implantation timepoints. Upon extraction on day 10 (left), hPSC-Heps encapsulated in sulfated alginate and empty sulfated alginate microbeads showed overall decreased PFO compared to hPSC-Hep-encapsulated and empty UP-LVG alginate microbeads (n >= 26 beads retrieved per group); similar but more pronounced difference was observed on day 20 (right) (n >= 9 beads retrieved per group).