ABSTRACT
Title
Adipose-derived stem cells for future cellular therapies in skeletal-muscle tissues regeneration
Authors
E. Arrigoni
Doctorate School in Pharmacological Science
Dept. of Medical Pharmacology, School of Medicine, Università degli Studi di Milano, Italy
Doctorate School in Pharmacological Science
Dept. of Medical Pharmacology, School of Medicine, Università degli Studi di Milano, Italy
Abstract
Every year several patients have to deal with bone tissue loss due to surgery, diseases or trauma. Bone tissue engineering aims to restore or repair musculoskeletal disorders through the development of bio-substitutes that requires the use of cells and scaffolds which should possess either adequate mechanical properties and interconnecting pores to allow cellular infiltration and vascularisation [1]. Adipose tissue represents a source of stem cells (ASCs) that can be easily isolated and that thank to their multi-differentiative potential [2] may represent a promising approach for bone regeneration. However there are several aspects that need to be faced before moving to the clinic: 1. the ability of human ASCs (hASCs) to osteo-differentiate may be affected by the donor’s physiological and pathological conditions 2. the use of selected pharmacological treatment may enhance the cellular plasticity 3. the design of preclinical models suitable to evaluate either the in vivo cytotoxicity and the use of a cellular therapy.
We have previously reported that age [3] and pathological conditions such as obesity affect the ability of hASCs to osteo-differentiate suggesting that not all donors may be an ideal sources of hASCs, and, since hASCs are known to be low immunogenic, we could consider either to apply allogenic cells or to improve their differentiative ability. Following this idea, we have studied the effect of Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells [4,5], on these progenitor cells. 72 hrs treatment of 50nM Reversine improves the ability of hASCs to differentiate into osteoblast like-cells (+45% of alkaline phosphatase activity), smooth muscle cells (+89% of α-actin expression) and skeletal muscle cells (myotubes formation of hASCs in co-culture with murine C2C12 cells) compared to control hASCs (72 hrs treated cells with vehicle - 0.05% DMSO).
Next, we have evaluated the ability of ASCs to regenerate a critical bone defect in a rabbit model. ASCs isolated from adipose tissue from interscapular region (rbASCs) were characterized and used, alone or in association with clinical-grade hydroxyapatite disks (HA), for autologous reimplantation in a critical bone defect in the proximal epiphysis of the tibia. 12 New Zealand rabbits were divided in 4 experimental groups: sham (just defect), just hydroxyapatite disk (HA) or just ASCs (ASCs) and ASCs pre-loaded on HA disks (ASCs-HA) [6,7]. Each rbASCs populations were analyzed in vitro for their ability to self-renew and to osteo-differentiate in the absence or in the presence of HA. 8 weeks after the implants, gross appearance, X-ray, histological and immunochemical analyses were performed showing that ASCs-HA bioconstructs are able to nicely heal the bone defects with the production of new formed mature bone tissue that also shows biomechanical properties similar to the native bone. Right now, we have just set a new experimental model with 7 minipigs: we would like to restore a critical osteochondral defect produced in the knees of these animas by autologous ASCs (pASCs) in association with porous photocrosslinked oligo [(polyethylene glycol) fumarate] (OPF) hydrogels.
We think that adipose-derived stem cells are an important cellular source that might be used for several research’s applications. Indeed, ASCs may give a significant impact in the regenerative medicine field and they are believed to be good candidates to screen drugs’ cytotoxicity and to study their mechanism of action.
We have previously reported that age [3] and pathological conditions such as obesity affect the ability of hASCs to osteo-differentiate suggesting that not all donors may be an ideal sources of hASCs, and, since hASCs are known to be low immunogenic, we could consider either to apply allogenic cells or to improve their differentiative ability. Following this idea, we have studied the effect of Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells [4,5], on these progenitor cells. 72 hrs treatment of 50nM Reversine improves the ability of hASCs to differentiate into osteoblast like-cells (+45% of alkaline phosphatase activity), smooth muscle cells (+89% of α-actin expression) and skeletal muscle cells (myotubes formation of hASCs in co-culture with murine C2C12 cells) compared to control hASCs (72 hrs treated cells with vehicle - 0.05% DMSO).
Next, we have evaluated the ability of ASCs to regenerate a critical bone defect in a rabbit model. ASCs isolated from adipose tissue from interscapular region (rbASCs) were characterized and used, alone or in association with clinical-grade hydroxyapatite disks (HA), for autologous reimplantation in a critical bone defect in the proximal epiphysis of the tibia. 12 New Zealand rabbits were divided in 4 experimental groups: sham (just defect), just hydroxyapatite disk (HA) or just ASCs (ASCs) and ASCs pre-loaded on HA disks (ASCs-HA) [6,7]. Each rbASCs populations were analyzed in vitro for their ability to self-renew and to osteo-differentiate in the absence or in the presence of HA. 8 weeks after the implants, gross appearance, X-ray, histological and immunochemical analyses were performed showing that ASCs-HA bioconstructs are able to nicely heal the bone defects with the production of new formed mature bone tissue that also shows biomechanical properties similar to the native bone. Right now, we have just set a new experimental model with 7 minipigs: we would like to restore a critical osteochondral defect produced in the knees of these animas by autologous ASCs (pASCs) in association with porous photocrosslinked oligo [(polyethylene glycol) fumarate] (OPF) hydrogels.
We think that adipose-derived stem cells are an important cellular source that might be used for several research’s applications. Indeed, ASCs may give a significant impact in the regenerative medicine field and they are believed to be good candidates to screen drugs’ cytotoxicity and to study their mechanism of action.
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