Tissue engineering a fetal membrane

Abstract

The aim of this study was to construct an artificial fetal membrane (FM) by combination of human amniotic epithelial stem cells (hAESCs) and a mechanically enhanced collagen scaffold containing encapsulated human amniotic stromal fibroblasts (hASFs). Such a tissue-engineered FM may have the potential to plug structural defects in the amniotic sac after antenatal interventions, or to prevent preterm premature rupture of the FM. The hAESCs and hASFs were isolated from human fetal amniotic membrane (AM). Magnetic cell sorting was used to enrich the hAESCs by positive ATP-binding cassette G2 selection. We investigated the use of a laminin/fibronectin (1:1)-coated compressed collagen gel as a novel scaffold to support the growth of hAESCs. A type I collagen gel was dehydrated to form a material mimicking the mechanical properties and ultra-structure of human AM. hAESCs successfully adhered to and formed a monolayer upon the biomimetic collagen scaffold. The resulting artificial membrane shared a high degree of similarity in cell morphology, protein expression profiles, and structure to normal fetal AM. This study provides the first line of evidence that a compacted collagen gel containing hASFs could adequately support hAESCs adhesion and differentiation to a degree that is comparable to the normal human fetal AM in terms of structure and maintenance of cell phenotype.

FIG. 1.

(A) Compacted collagen gel embedded with human amniotic stromal fibroblasts (hASFs); (B) denuded amniotic membrane (dAM); scanning electron microscopy (SEM) of (C) compacted collagen scaffold surface and (D) dAM surface. Scale bar (A, B) 5 mm and (C, D) 10 μm.

FIG. 2.

The rheological properties of compacted collagen containing hASFs and dAM also containing native hASFs (n=3). Preliminary oscillatory stress sweeps carried out at 37°C (A). Oscillatory frequency sweeps carried out at 37°C at a controlled stress of 5 Pa for both scaffolds. Closed and open circles refer to G′ (storage modulus) and G′′ (loss modulus), respectively (B). Color images available online at www.liebertonline.com/tea

FIG. 3.

Magnetically activated cell sorting to enrich human amniotic epithelial stem cells (hAESCs) (A). ABCG2-negative cells flow through the column and ABCG2-positive cells adhered on the column. The ABCG2-positive cells were flushed out by firmly pushing the plunger into the column after removing the magnets (B). The colony-forming efficiency of ABCG2-positive cell group (C) and ABCG2-negative cell group (D). Color images available online at www.liebertonline.com/tea

FIG. 4.

hAESCs cultured on noncoated (A), collagen IV-coated (B), fibronectin-coated (C), laminin-coated (D), and fibronectin/laminin (1:1)-coated (E) compressed gels. (F) normal AM. Scale bar=50 μm.

FIG. 5.

SEM of hAESCs grown on fibronectin/laminin (1:1)-coated compressed collagen scaffold (A). Control normal human AM epithelial cells (B). Scale bar=20 μm.

FIG. 6.

Hematoxylin and eosin staining of tissue-engineered human AM (A) and normal (native) human AM (B). Scale bar=50 μm. Color images available online at www.liebertonline.com/tea

FIG. 7.

Transmission electron microscopy images of tissue engineering AM and normal AM. (A) Basal cells appeared to adhere well to the compressed collagen scaffold via hemidesmosome attachments (arrows). (B) Neighboring cells displayed desmosome junctions (arrows). (C) Hemidesmosome attachments in normal AM (arrows). (D) Desmosome junctions in normal AM (arrows). Scale bars=1 μm.

FIG. 8.

Immunofluorescent staining for the hAESC markers ABCG2, CK19, and propidium iodide costained nuclei. Artificial AM stained for ABCG2 [see the merged color images (C) for ABCG2 (green; B) and propidium iodide (red; A)] and CK19 [see the merged color images (I) for CK19 (green; H) and propidium iodide (red; G)]. Normal AM-stained ABCG2 [see the merged color images (F) for ABCG2 (green; E) and propidium iodide (red; D)] and CK19 [see the merged color images (L) for CK19 (green; K) and propidium iodide (red; J)]. Scale bar=50 μm. Color images available online at www.liebertonline.com/tea