New adipose tissue formation by human adipose-derived stem cells with hyaluronic acid gel in immunodeficient mice

Abstract

Background: Currently available injectable fillers have demonstrated limited durability. This report proposes the in vitro culture of human adipose-derived stem cells (hASCs) on hyaluronic acid (HA) gel for in vivo growth of de novo adipose tissue.

Methods: For in vitro studies, hASCs were isolated from human adipose tissue and were confirmed by multi-lineage differentiation and flow cytometry. hASCs were cultured on HA gel. The effectiveness of cell attachment and proliferation on HA gel was surveyed by inverted light microscopy. For in vivo studies, HA gel containing hASCs, hASCs without HA gel, HA gel alone were allocated and subcutaneously injected into the subcutaneous pocket in the back of nude mice (n=6) in each group. At eight weeks post-injection, the implants were harvested for histological examination by hematoxylin and eosin (H&E) stain, Oil-Red O stain and immunohistochemical staining. The human-specific Alu gene was examined.

Results: hASCs were well attachment and proliferation on the HA gel. In vivo grafts showed well-organized new adipose tissue on the HA gel by histologic examination and Oil-Red O stain. Analysis of neo-adipose tissues by PCR revealed the presence of the Alu gene. This study demonstrated not only the successful culture of hASCs on HA gel, but also their full proliferation and differentiation into adipose tissue.

Conclusions: The efficacy of injected filler could be permanent since the reduction of the volume of the HA gel after bioabsorption could be replaced by new adipose tissue generated by hASCs. This is a promising approach for developing long lasting soft tissue filler.

Keywords: adipose tissue; human adipose-derived stem cells.

Fig 1

The hASCs were well-differentiated into three cell lineages. Lipid vacuole formation after 2 weeks culture of hASCs in adipogenic medium (upper row, left). Positive Oil-Red O stain (X200) (upper row, middle). Positive Alcian Blue stain after 2-week culture of hASCs in chondrogenic medium (X200) (upper row, right). Positive von Kossa stain after 4-week culture of hASCs in osteogenic medium (X200) (middle row, left). Flow cytometric analysis on hASCs for the expression of CD34, CD45, CD44, CD90, and CD105 was performed (Red). Cells stained with Isotype control IgG were examined as a control (black).

Fig 2

Inverted light microscope image showing hASCs seeding in HA gel. hASCs proliferate and aggregate in HA gel on Day 1,3,7 (X100) (red color indicated hASCs with CM-DiI stain). An MTT assay was used to check hASCs cell proliferation in HA gel. No significantly difference between HA-hASCs and hASCs alone group in MTT assay. Error bars indicated deviation.

Fig 3

Macroscopic appearance of grafts in nude mice. Suspension of HA gel containing hASC was subcutaneously injected into the back of each nude mouse. (Left) New adipose tissue formation was noted near the residual HA gel at eight weeks after injection. (Right) In the control group, the residual clear HA gel was noted in subcutaneous areas. The ruler represents 2 cm.

Fig 4

Histological examination of the grafts stained by hematoxylin-eosin stain eight weeks after injection of suspension of HA gel with hASCs revealed newly formed adipose tissue. The black stars (*) indicate adipose tissue (scale bar 50μm). Oil-Red O stain (red arrows) revealed the gradual replacement of degraded HA gel with newly formed adipose tissue (red stain) (scale bar 100μm). In the immunohistochemical stain showed CD44 positive cell marker and CM-DiI positive in newly adipose tissue (scale bar 50μm, 100μm respectively).

Fig 5

Results of PCR analysis of Alu gene in graft specimens. 1 was HA gel with hASCs graft. 2 was HA gel only. 3 was hASCs only. 4 was HA gel with medium. 5 was control group. P was positive control.