Human Adipose-derived Stem/stromal Cells Research Articles

Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells.

Dysfunctions in adipose tissue cells are responsible for several obesity-related metabolic diseases. Understanding the process of adipocyte formation is thus fundamental for understanding these diseases. The adipocyte differentiation of adipose-derived stem/stromal cells (ADSCs) showed a reduction in the mRNA level of the interleukin 21 receptor (IL21R) during this process. Although the receptor has been associated with metabolic diseases, few studies have examined its function in stem cells. In this study, we used confocal immunofluorescence assays to determine that IL21R colocalizes with mitochondrial protein ATP5B, ALDH4A1, and the nucleus of human ADSCs. We demonstrated that silencing and overexpression of IL21R did not affect the cell proliferation and mitochondrial activity of ADSCs. However, IL21R silencing did reduce ADSC adipogenic capacity. Further studies are needed to understand the mechanism involved between IL21R and the adipogenic differentiation process.

Anti-DKK1 Enhances the Early Osteogenic Differentiation of Human Adipose-Derived Stem/Stromal Cells.

Adipose-derived stem/stromal cells (ASCs) have been previously used for bone repair. However, significant cell heterogeneity exists within the ASC population, which has the potential to result in unreliable bone tissue formation and/or low efficacy. Although the use of cell sorting to lower cell heterogeneity is one method to improve bone formation, this is a technically sophisticated and costly process. In this study, we tried to find a simpler and more deployable solution-blocking antiosteogenic molecule Dickkopf-1 (DKK1) to improve osteogenic differentiation. Human adipose-derived stem cells were derived from = 5 samples of human lipoaspirate. In vitro, anti-DKK1 treatment, but not anti-sclerostin (SOST), promoted ASC osteogenic differentiation, assessed by alizarin red staining and real-time polymerase chain reaction (qPCR). Increased canonical Wnt signaling was confirmed after anti-DKK1 treatment. Expression levels of DKK1 peaked during early osteogenic differentiation (day 3). Concordantly, anti-DKK1 supplemented early (day 3 or before), but not later (day 7) during osteogenic differentiation positively regulated osteoblast formation. Finally, anti-DKK1 led to increased transcript abundance of the Wnt inhibitor SOST, potentially representing a compensatory cellular mechanism. In sum, DKK1 represents a targetable "molecular brake" on the osteogenic differentiation of human ASC. Moreover, release of this brake by neutralizing anti-DKK1 antibody treatment at least partially rescues the poor bone-forming efficacy of ASC.

Investigating the Effects of Tissue-Specific Extracellular Matrix on the Adipogenic and Osteogenic Differentiation of Human Adipose-Derived Stromal Cells Within Composite Hydrogel Scaffolds.

While it has been postulated that tissue-specific bioscaffolds derived from the extracellular matrix (ECM) can direct stem cell differentiation, systematic comparisons of multiple ECM sources are needed to more fully assess the benefits of incorporating tissue-specific ECM in stem cell culture and delivery platforms. To probe the effects of ECM sourced from decellularized adipose tissue (DAT) or decellularized trabecular bone (DTB) on the adipogenic and osteogenic differentiation of human adipose-derived stem/stromal cells (ASCs), a novel detergent-free decellularization protocol was developed for bovine trabecular bone that complemented our established detergent-free decellularization protocol for human adipose tissue and did not require specialized equipment or prolonged incubation times. Immunohistochemical and biochemical characterization revealed enhanced sulphated glycosaminoglycan content in the DTB, while the DAT contained higher levels of collagen IV, collagen VI and laminin. To generate platforms with similar structural and biomechanical properties to enable assessment of the compositional effects of the ECM on ASC differentiation, micronized DAT and DTB were encapsulated with human ASCs within methacrylated chondroitin sulfate (MCS) hydrogels through UV-initiated crosslinking. High ASC viability (>90%) was observed over 14 days in culture. Adipogenic differentiation was enhanced in the MCS+DAT composites relative to the MCS+DTB composites and MCS controls after 14 days of culture in adipogenic medium. Osteogenic differentiation studies revealed a peak in alkaline phosphatase (ALP) enzyme activity at 7 days in the MCS+DTB group cultured in osteogenic medium, suggesting that the DTB had bioactive effects on osteogenic protein expression. Overall, the current study suggests that tissue-specific ECM sourced from DAT or DTB can act synergistically with soluble differentiation factors to enhance the lineage-specific differentiation of human ASCs within 3-D hydrogel systems.

Peptide-modified methacrylated glycol chitosan hydrogels as a cell-viability supporting pro-angiogenic cell delivery platform for human adipose-derived stem/stromal cells.

Cell-based therapies involving the injection of adipose-derived stem/stromal cells (ASCs) within rationally designed biomaterials are a promising approach for stimulating angiogenesis. With this focus, the current work explored the effects of incorporating integrin-binding RGD or IKVAV peptides within in situ-gelling N-methacrylate glycol chitosan (MGC) hydrogels on the response of encapsulated human ASCs. Initial studies focused on hydrogel characterization to validate that the MGC, MGC-RGD, and MGC-IKVAV hydrogels had similar biomechanical properties. ASC viability following encapsulation and culture under 2% O2 was significantly impaired in the MGC-IKVAV group relative to the MGC and MGC-RGD groups. In contrast, sustained viability, along with enhanced cell spreading and metabolic activity were observed in the MGC-RGD group. Investigation of angiogenic transcription suggested that the incorporation of the peptide groups did not substantially alter the pro-angiogenic gene expression profile of the encapsulated ASCs after 7 days of culture under 2% O2. Consistent with the in vitro findings, preliminary in vivo characterization following subcutaneous implantation into NOD/SCID mice showed that ASC retention was enhanced in the MGC-RGD hydrogels relative to the MGC-IKVAV group at 14 days. Further, the encapsulated ASCs in the MGC and MGC-RGD groups promoted murine CD31+ endothelial cell recruitment to the peri-implant region. Overall, the results indicate that the MGC-RGD and MGC hydrogels are promising platforms for ASC delivery, and suggest that strategies that support long-term ASC viability can augment in vivo angiogenesis through paracrine mechanisms. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 571-585, 2019.

Mechanically resilient injectable scaffolds for intramuscular stem cell delivery and cytokine release

A promising strategy for treating peripheral ischemia involves the delivery of stem cells to promote angiogenesis through paracrine signaling. Treatment success depends on cell localization, retention, and survival within the mechanically dynamic intramuscular (IM) environment. Herein we describe an injectable, in situ-gelling hydrogel for the IM delivery of adipose-derived stem/stromal cells (ASCs), specifically designed to withstand the dynamic loading conditions of the lower limb and facilitate cytokine release from encapsulated cells. Copolymers of poly(trimethylene carbonate)-b-poly(ethylene glycol)-b-poly(trimethylene carbonate) diacrylate were used to modulate the properties of methacrylated glycol chitosan hydrogels crosslinked by thermally-initiated polymerization using ammonium persulfate and N,N,N′,N′-tetramethylethylenediamine. The scaffolds had an ultimate compressive strain over 75% and maintained mechanical properties during compressive fatigue testing at physiological levels. Rapid crosslinking (<3 min) was achieved at low initiator concentration (5 mM). Following injection and crosslinking within the scaffolds, human ASCs demonstrated high viability (>90%) over two weeks in culture under both normoxia and hypoxia. Release of angiogenic and chemotactic cytokines was enhanced from encapsulated cells under sustained hypoxia, in comparison to normoxic and tissue culture polystyrene controls. When delivered by IM injection in a mouse model of hindlimb ischemia, human ASCs were well retained in the scaffold over 28 days and significantly increased the IM vascular density compared to untreated controls.

An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells

For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 106 ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20–100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo.

Decellularized adipose tissue microcarriers as a dynamic culture platform for human adipose-derived stem/stromal cell expansion.

With the goal of designing a clinically-relevant expansion strategy for human adipose-derived stem/stromal cells (ASCs), methods were developed to synthesize porous microcarriers derived purely from human decellularized adipose tissue (DAT). An electrospraying approach was applied to generate spherical DAT microcarriers with an average diameter of 428±41μm, which were soft, compliant, and stable in long-term culture without chemical crosslinking. Human ASCs demonstrated enhanced proliferation on the DAT microcarriers relative to commercially-sourced Cultispher-S microcarriers within a spinner culture system over 1 month. ASC immunophenotype was maintained post expansion, with a trend for reduced expression of the cell adhesion receptors CD73, CD105, and CD29 under dynamic conditions. Upregulation of the early lineage-specific genes PPARγ, LPL, and COMP was observed in the ASCs expanded on the DAT microcarriers, but the cells retained their multilineage differentiation capacity. Comparison of adipogenic and osteogenic differentiation in 2-D cultures prepared with ASCs pre-expanded on the DAT microcarriers or Cultispher-S microcarriers revealed similar adipogenic and enhanced osteogenic marker expression in the DAT microcarrier group, which had undergone a higher population fold change. Further, histological staining results suggested a more homogeneous differentiation response in the ASCs expanded on the DAT microcarriers as compared to either Cultispher-S microcarriers or tissue culture polystyrene. A pilot chondrogenesis study revealed higher levels of chondrogenic gene and protein expression in the ASCs expanded on the DAT microcarriers relative to all other groups, including the baseline controls. Overall, this study demonstrates the promise of applying dynamic culture with tissue-specific DAT microcarriers as a means of deriving regenerative cell populations.

Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments

Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications. This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.

Influence of Donor Age and Passage Number on Angiogenic Activity in Human Adipose-Derived Stem Cell-Conditioned Media

Introduction: Adipose-derived stem/stromal cells (ASCs) are considered a promising cell source for therapeutic angiogenesis because the cells can be prepared following a minimally invasive procedure and because they secrete a variety of angiogenic cytokines. In the present study, the influence of donor age and passage number on angiogenic activity of ASC-conditioned media (ASC-CM) was examined. Methods: Human ASCs (donor age, 5 months to 82 years; n = 10) were cultured, and ASC-CM were collected at passages 2, 4, and 6. The angiogenic activity of ASC-CM was evaluated with the tube formation assay using a system in which human umbilical vein endothelial cells (HUVECs) and fibroblasts were co-cultured. The concentrations of vascular endothelial growth factor-A (VEGF-A) and hepatocyte growth factor (HGF) in each ASC-CM were measured using an enzyme-linked immunosorbent assay. Results: A donor age over 60 years affected the proliferative capacity of ASCs at passage 4 and later. ASCCM significantly enhanced HUVEC tube formation, and this response was not influenced by donor age. ASC-CM at passage 6 showed a lower tube formation capacity compared to ASC-CM at passage 4, although the capacity was still equivalent to the positive control (medium containing10 ng/mL VEGF-A). A donor age over 26 years affected VEGF-A but not HGF levels in ASC-CM, although we found no direct correlation between VEGF-A/HGF levels and the tube formation capacity. Conclusion: Our results demonstrate a passage number-dependent but a donor age-independent decline in the angiogenic activity of ASC-CM.

Mesenchymal Stem Cells from Bichat's Fat Pad:In VitroComparison with Adipose-Derived Stem Cells from Subcutaneous Tissue

Adipose-derived stem/stromal cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Since Bichat's fat pad is easily accessible for dentists and maxillo-facial surgeons, we compared the features of ASCs from Bichat's fat pad (BFP-ASCs) with human ASCs from subcutaneous adipose tissue (SC-ASCs). BFP-ASCs isolated from a small amount of tissue were characterized for their stemness and multidifferentiative ability. They showed an important clonogenic ability and the typical mesenchymal stem cell immunophenotype. Moreover, when properly induced, osteogenic and adipogenic differentiation markers, such as alkaline phosphatase activity, collagen deposition and lipid vacuoles formation, were promptly observed. Growth of both BFP-ASCs and SC-ASCs in the presence of human serum and their adhesion to natural and synthetic scaffolds were also assessed. Both types of ASCs adapted rapidly to human autologous or heterologous sera, increasing their proliferation rate compared to standard culture condition, and all the cells adhered finely to bone, periodontal ligament, collagen membrane, and polyglycol acid filaments that are present in the oral cavity or are commonly used in oral surgery. At last, we showed that amelogenin seems to be an early osteoinductive factor for BFP-ASCs, but not SC-ASCs, in vitro. We conclude that Bichat's fat pad contains BFP-ASCs with stemness features that are able to differentiate and adhere to biological supports and synthetic materials. They are also able to proliferate in the presence of human serum. For all these reasons we propose BFP-ASCs for future therapies of periodontal defects and bone regeneration.