Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells

Abstract

Background: Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations.

Purpose: To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation.

Study design: Controlled laboratory study.

Methods: MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated.

Results: The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics-(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)-and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 10⁶ ± 3.7 × 10⁵ and 1.6 × 10⁶ ± 4.7 × 10⁵ per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P < .0001), and collagen type 2 ( P = .044) gene expression compared with hADSCs, respectively, after culture in differentiation medium. Moreover, both hAECs and hAMSCs produced significantly greater quantities of mineralized ( P < .0001) and cartilaginous ( P = .0004) matrix at earlier time points compared with hADSCs when cultured under identical osteogenic and chondrogenic differentiation conditions, respectively.

Conclusion: Amnion-derived MSCs demonstrate a greater differentiation potential toward bone and cartilage compared with hADSCs.

Clinical relevance: Amniotic MSCs may be the source of choice in the regenerative treatment of bone or osteochondral musculoskeletal disease. They show significantly higher yields and better differentiation toward these tissues than MSCs derived from adipose.

Keywords: adipose; bone; cartilage; differentiation; orthopaedics; perinatal; regenerative medicine; stromal cell.

Figure 1

Amniotic membrane harvest, mesenchymal stromal cell (MSC) isolation, and characterization. (A) Representative image of a human placenta with umbilical cord (black arrowhead) and epithelial layer of the amniotic membrane (white arrowhead) facing upward. (B) Representative images depicting the separation of the amniotic membrane (white arrowhead) from the chorion (black arrowhead). Representative hematoxylin and eosin (H&E) histological sections: (C) fresh amniotic membrane exhibiting a continuous layer of human amniotic epithelial cells (hAECs) (arrowheads) and intact stroma containing human amniotic mesenchymal stromal cells (hAMSCs) (box), (D) amniotic membrane after incomplete removal of hAECs (arrowheads) by use of 0.125% trypsin yielding a “mixed” population of amniotic MSCs, and (E) amniotic membrane after complete removal of hAECs by use of 0.25% trypsin. Polarized light microscopic images of monolayer culture expanded (passage 2) amnion cells: (F) enriched hAECs (cuboidal morphology—black arrowheads), (G) a mixed population of hAECs (black arrowheads) and hAMSCs (white arrowheads) after incomplete dissociation of hAECs, and (H) enriched hAMSCs (spindle morphology—white arrowheads). (I) Graph of average viable cell yields from term human amniotic membranes. (J) Flow cytometric analysis of enriched hAECs, mixed, and enriched hAMSCs illustrated positive expression for MSC markers (CD73, CD90, and CD105) and the absence of the hematolymphocyte common antigen, CD45. Additionally, enriched hAECs were positive for the epithelial marker, EpCAM, while enriched hAMSCs further confirmed their identity (data represented as percentage of cells with positive staining ± SEM).

Figure 2

Osteogenic gene transcript expression of human amnion-derived mesenchymal stromal cells and epithelial cells (hAMSCs and hAECs) and human adipose-derived stromal cells (hADSCs). Gene transcript expression levels of (A) runx-2 and (B) osteocalcin (OCN) in hAEC, mixed (hAEC + hAMSC co-culture), and hAMSC groups relative to hADSCs (dotted line) cultured in basal medium. (C) Runx-2 and (D) osteocalcin expression of amnion-derived mesenchymal stromal cell groups cultured in osteogenic differentiation medium compared with respective expression levels in hADSCs cultured at the same time points (n = 4 per group per time point). Lines indicate statistical difference (P < .05) between connected groups. *Statistical differences compared with the hADSC group at the same time point (P < .05).

Figure 3

Production of mineralized matrix in human amnion-derived mesenchymal stromal cells and epithelial cells (hAMSCs and hAECs) and human adipose-derived stromal cells (hADSCs). (A) Alizarin Red staining of monolayer cultures of hAECs, mixed (hAEC + hAMSC co-culture), hAMSCs, and hADSCs cultured in osteogenic differentiation medium (red = positive staining for mineralization) at day 14 and 28 (n = 3 per group per time point). (B) Graph of percentage area of the well that stained positive for mineralization. Alizarin Red staining of (C) hAECs cultured in basal medium for 28 days. *Statistical differences compared with the hADSC group at the same time point (P < .05). ^#Statistical difference (P < .05) within each cell group compared with its respective day 14 value.

Figure 4

Chondrogenic gene transcript expression of human amnion-derived mesenchymal stromal cells and epithelial cells (hAMSCs and hAECs) and human adipose-derived stromal cells (hADSCs). Transcript expression levels of (A) Sox-9 and (B) aggrecan in hAEC, mixed (hAEC + hAMSC co-cultured), and hAMSC groups relative to hADSCs (dotted line) cultured in basal medium. (C) Sox-9, (D) aggrecan, and (E) runx-2 expression of amnion mesenchymal stromal cell groups cultured in chondrogenic differentiation medium compared with respective expression levels in hADSCs cultured at the same time points (n = 4 per group per time point). Lines indicate statistical difference (P < .05) between connected groups. *Statistical differences compared with the hADSC group at the same time point (P < .05).

Figure 5

Collagen type 2 gene transcript and matrix deposition in human amnion-derived mesenchymal stromal cells and epithelial cells (hAMSCs and hAECs) and human adipose-derived stromal cells (hADSCs). (A) Immunohistochemical staining (brown = positive, blue = nuclei) for collagen type 2 in hAMSC, mixed (hAEC + hAMSC co-culture), and hADSC cell pellets cultured in chondrogenic differentiation medium. (B) Gene transcript expression levels of collagen type 2 in mixed (hAEC + hAMSC co-culture) and hAMSC groups relative to hADSCs (dotted line) cultured in basal medium. (C) Collagen type 2 expression of amnion-derived mesenchymal stromal cells cultured in chondrogenic differentiation medium compared with respective expression in hADSCs cultured at the same time points. Lines indicate statistical difference (P < .05) between connected groups. *Statistical differences compared with the hADSC group at the same time point (P < .05).

Figure 6

Glycosaminoglycan (GAG) content in pellets of human amnion-derived mesenchymal stromal cells and epithelial cells (hAMSCs and hAECs) and human adipose-derived stromal cells (hADSCs). (A) Alcian Blue staining for glycosaminoglycan of hAEC, mixed (hAEC + hAMSC co-culture), hAMSC, and hADSC cell pellets (blue = positive staining) cultured in chondrogenic differentiation medium at day 3 and day 7. (B) Quantitative analysis of percentage area of the cell pellet stained positive for GAG and (C) comparison of hAEC, mixed (hAEC + hAMSC co-culture), hAMSC, and hADSC cell pellet diameter cultured in differentiation medium at day 3 and 7. *Statistical differences compared with the hADSC group at the same time point (P < .05). ^#Statistical difference (P < .05) within each cell group compared with its respective day 3 value.