Culture Adipose-derived Stem Cells Research Articles

Lactoferrin Mediates Enhanced Osteogenesis of Adipose-Derived Stem Cells: Innovative Molecular and Cellular Therapy for Bone Repair.

A prospective source of stem cells for bone tissue engineering is adipose-derived stem cells (ADSCs), and BMP-2 has been proven to be highly effective in promoting the osteogenic differentiation of stem cells. Rarely has research been conducted on the impact of lactoferrin (LF) on ADSCs' osteogenic differentiation. As such, in this study, we examined the effects of LF and BMP-2 to assess the ability of LF to stimulate ADSCs' osteogenic differentiation. The osteogenic medium was supplemented with the LF at the following concentrations to culture ADSCs: 0, 10, 20, 50, 100, and 500 μg/mL. The Cell Counting Kit-8 (CCK-8) assay was used to measure the proliferation of ADSCs. Calcium deposition, alkaline phosphatase (ALP) staining, real-time polymerase chain reaction (RT-PCR), and an ALP activity assay were used to establish osteogenic differentiation. RNA sequencing analysis was carried out to investigate the mechanism of LF boosting the osteogenic development of ADSCs. In the concentration range of 0-100 μg/mL, LF concentration-dependently increased the proliferative vitality and osteogenic differentiation of ADSCs. At a dose of 500 μg/mL, LF sped up and enhanced differentiation, but inhibited ADSCs from proliferating. LF (100 and 500 μg/mL) produced more substantial osteoinductive effects than BMP-2. The PI3 kinase/AKT (PI3K/AKT) and IGF-R1 signaling pathways were significantly activated in LF-treated ADSCs. The in vitro study results showed that LF could effectively promote osteogenic differentiation of ADSCs by activating the PI3K/AKT and IGF-R1 pathways. In our in vitro investigation, an LF concentration of 100 μg/mL was optimal for osteoinduction and proliferation. Our study suggests that LF is an attractive alternative to BMP-2 in bone tissue engineering. As a bioactive molecule capable of inducing adipose stem cells to form osteoblasts, LF is expected to be clinically used in combination with biomaterials as an innovative molecular and cellular therapy to promote bone repair.

Culturing Adipose-Derived Stem Cells Under Serum-Free Conditions.

Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave very different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.

Rapid Stem Cell Extraction and Culture Device for Regenerative Therapy Using Biodegradable Nonwoven Fabrics with Strongly Oriented Fibers (Adv. Mater. Interfaces 16/2022)

Biodegradable Nonwoven Fabrics In article number 2101776, Hiroshi Sunami and co-workers develop biodegradable nonwoven fabrics that can be used to extract and culture adipose-derived stem cells (ADSCs) from adipose tissue slices without enzyme digestions. ADSCs and ADSC sheets for regenerative therapies are produced more rapidly by cultivating cells on the nonwoven fabrics.

Constructing Tissue-Engineered Dressing Membranes with Adipose-Derived Stem Cells and Acellular Dermal Matrix for Diabetic Wound Healing: A Comparative Study of Hypoxia- or Normoxia-Culture Modes.

Diabetes foot ulcer (DFU) is a serious complication of diabetes, characterized by impaired vascular function, limited angiogenesis, and chronic inflammation. Direct stem cell injection on treating DFU is far from satisfactory in clinical practice, as this therapy neither protects nor localizes the injected cell suspension at the chronic ulcer site. Meanwhile, most of injected cells gradually perished within several days due to senescence or apoptosis. Acellular dermal matrix (ADM) has the potential to act as excellent cell delivery vehicles, considering it is highly biomimetic to native dermal tissue, has low immunogenicity, and suitable for stem cell attachment and proliferation. Hypoxia culture has significantly enhanced effects on the survival ability of in vitro cultured stem cells, indicating this culture mode is a suitable way for inhibiting the senescence or apoptosis of transplanted cells. In the current study, we, respectively, culture adipose-derived stem cells (ADSCs) on an ADM membrane under a hypoxia or normoxia condition to construct two kinds of tissue-engineered dressing membranes (H-ADSCs/ADM and N-ADSCs/ADM) and then comparatively evaluated their efficacy on DFU healing using a diabetic rat model. In vitro results showed that hypoxia precondition could stimulate the ADSCs secreting VEGF-A, and the culture medium from hypoxia-preconditioned ADSCs could enhance the proliferation, migration, and angiogenesis of HUVECs. In vivo results indicated that compared to the N-ADSCs/ADM membrane, the transplanted cells in the H-ADSCs/ADM membrane can survive longer at the chronic ulcer site, consequently improve angiogenesis, inhibit inflammation, and increase extracellular matrix remodeling, eventually accelerating DFU closure. This study provides an innovative covering graft for the treatment of DFU in the clinic.

Rapid Stem Cell Extraction and Culture Device for Regenerative Therapy Using Biodegradable Nonwoven Fabrics with Strongly Oriented Fibers

AbstractBiodegradable nonwoven fabrics that can be used to extract and culture adipose‐derived stem cells (ADSCs) from adipose tissue slices without enzyme digestion are developed. The fabrics are made of fibers consisting of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) and hydroxyapatite (HAp), which can be strongly oriented by adjusting the fiber deposition conditions. ADSCs are extracted from adipose tissue slices and cultured, and thick cell sheets are produced by placing nonwoven fabric on the adipose tissue slices or sandwiching the adipose tissue slices between two layers of fabric. Nonwoven fabrics with large anisotropy extract and culture ADSCs from adipose tissue slices more quickly than those with small anisotropy. In addition, cell sheets are produced more rapidly by cultivating cells on a culture vessel with low‐cell adhesion. Finally, it confirms that ADSCs extracted and cultured from adipose tissue slices using nonwoven fabrics maintain the characteristics of mesenchymal stem cells.

Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells

Chronic non-healing wounds are detrimental for the quality of life of the affected individuals and represent a major burden for the health care systems. Adipose-derived stem cells (ASCs) are being investigated for the development of novel treatments of chronic wounds, as they have shown several positive effects on wound healing. While these effects appear to be mediated by the release of soluble factors, it is has also become apparent that the extracellular matrix (ECM) deposited by ASCs is essential in several phases of the wound healing process. In this work, we describe an approach to produce ECM scaffolds derived from ASCs in culture. Upon growth of ASCs into an overconfluent cell layer, a detergent-based cell extraction approach is applied to remove the cellular components. The extraction is followed by an enzymatic treatment to remove the residual DNA. The resultant cell-derived scaffolds are depleted of cellular components, display low DNA remnant, and retain the native fibrillar organization of the ECM. Analysis of the molecular composition of the ECM scaffolds revealed that they are composed of collagens type I and III, and fibronectin. The decellularized scaffolds represent a substrate that supports adhesion and proliferation of primary human fibroblasts and dermal microvascular endothelial cells, indicating their potential as platforms for wound healing studies.

Physioxia: a more effective approach for culturing human adipose-derived stem cells for cell transplantation

BackgroundAlthough typically cultured at an atmospheric oxygen concentration (20–21%), adipose-derived stem cells (ASCs) reside under considerable low oxygen tension (physioxia) in vivo. In the present study, we explored whether and how physioxia could be a more effective strategy for culturing ASCs for transplantation.MethodsAfter isolation, human ASCs were cultured under physioxia (2% O2) and hyperoxia (20% O2) until assayed. WST-8, Transwell, tube formation, β-galactosidase staining, and annexin V-FITC/PI assays were used to evaluate cell proliferation, migration, angiogenesis, senescence, and apoptosis, respectively. Survivability was determined by an ischemia model in vitro and nude mouse model in vivo, and the underlying metabolic alterations were investigated by fluorescence staining, flow cytometry, and real-time polymerase chain reaction.ResultsCompared with those in the hyperoxia group, cells in the physioxia group exhibited increased proliferation, migration, and angiogenesis, and decreased senescence and apoptosis. The increased survival rate of ASCs cultured in physioxia was found both in ischemia model in vitro and in vivo. The underlying metabolic reprogramming was also monitored and showed decreased mitochondrial mass, alkalized intracellular pH, and increased glucose uptake and glycogen synthesis.ConclusionsThese results suggest that physioxia is a more effective environment in which to culture ASCs for transplantation owing to the maintenance of native bioactivities without injury by hyperoxia.

Bioactivity and composition of a preserved connective tissue matrix derived from human placental tissue

There are a wide variety of extracellular matrices that can be used for regenerative purposes. Placental tissue‐based matrices are quickly becoming an attractive option given the availability of the tissue source and the wide variety of bioactive molecules knows to exist in unprocessed placental tissues. As fresh placental tissues are seldom an option at the point of care, we examined both the composition and bioactivity of a commercially packaged flowable placental connective tissue matrix (FPTM) (BioECM®, Skye Biologics, Inc.) that was preserved by the proprietary HydraTek® process. The FPTM contained significant amounts of collagen and various growth factors such as bFGF, EGF, PDGF, KGF, and PIGF. In addition, it contained high levels of tissue inhibitors of metalloproteinases (TIMP‐1 and 2) and molecules known to modulate the immune response including TGF‐β and IL‐4. In terms of its bioactivity, the FPTM displayed the ability (1) to suppress INF‐γ secretion in activated T‐cells nearly fourfold over control media, (2) to inhibit methicillin resistant Staphylococcus aureus (MRSA) and Staphylococcus saprophyticus proliferation, (3) to increase the migration of adipose‐derived stem cells (ASCs) nearly threefold over control media and (4) to adhere to ASCs in culture. When ASCs were exposed to FPTM in culture, the cells maintained healthy morphology and showed no significant changes in the expression of five genes involved in tissue growth and repair as compared to culture in standard growth media. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2731–2740, 2018.

Growing Adipose-Derived Stem Cells Under Serum-Free Conditions.

Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave significantly different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.

Adipose-derived stem cells: A novel source of parathyroid cells for treatment of hypoparathyroidism

Hypoparathyroidism is characterized by decreased function of the parathyroid glands with underproduction of parathyroid hormone (PTH), which can lead to low levels of calcium in the blood, often causing cramping and twitching of muscles or tetany, and several other symptoms. Severe hypocalcemia is a life-threatening condition. At present, both medical and surgical treatments are offered to improve the blood calcium, but they are not a cure. Adipose-derived stem cells (ADSCs), derived from the adipose tissue, are confirmed to be multipotent with adipogenic, chondrogenic, neurogenic, myogenic and osteogenic capabilities. Our hypothesis is that human ADSCs in culture can be differentiated into parathyroid cells, and used to reconstitute function.

Allogeneic Platelet Releasate Preparations Derived via a Novel Rapid Thrombin Activation Process Promote Rapid Growth and Increased BMP-2 and BMP-4 Expression in Human Adipose-Derived Stem Cells.

The administration of human adipose-derived stem cells (ASCs) represents a promising regenerative therapy for the treatment of orthopedic injuries. While ASCs can be easily isolated from liposuction-derived adipose tissue, most clinical applications will likely require in vitro culture expansion of these cells using nonxenogeneic components. In this study, platelet releasate was generated using a novel rapid thrombin activation method (tPR). ASCs grown in media supplemented with tPR proliferated much faster than ASCs grown in media supplemented with 10% fetal bovine serum. The cells also retained the ability to differentiate along chondrogenic, adipogenic, and osteogenic lineages. The tPR cultured ASCs displayed elevated expression of BMP-4 (5.7 ± 0.97-fold increase) and BMP-2 (4.7 ± 1.3-fold increase) and decreased expression of PDGF-B (4.0 ± 1.4-fold decrease) and FGF-2 (33 ± 9.0-fold decrease). No significant changes in expression were seen with TGF-β and VEGF. This pattern of gene expression was consistent across different allogeneic tPR samples and different ASC lines. The use of allogeneic rapidly activated tPR to culture ASCs is associated with both an increased cell yield and a defined gene expression profile making it an attractive option for cell expansion prior to cell-based therapy for orthopedic applications.

Plasticity and banking potential of cultured adipose tissue derived mesenchymal stem cells

The present day research on stem cells is yet not filled to the gunwales. The correlation of stem cell technology with tissue repair still has a long way to go. Since Embryonic stem cells are a kind of thorn inside when it comes to therapeutics, there emerged few potent contemporary sources of stem cells. Though bone marrow proves to be the pioneer among these, they lose themselves to adipose tissue in various aspects. The major shortcoming of bone marrow lies in lieu of its loss in potency with age. Adipose tissue puts up a tough competition among leading edge stem cell sources like cord blood and cord matrix. Adipose tissue wins over its counterparts in that it possesses astounding proliferation potency in vitro and holds a prominent stand in showcasing in vivo tissue repair efficacy. In spite of its precedence, the whole enchilada of adipose derived stem cells is still in its salad days. In our work we aim at excogitating the Mesenchymal stem cell population present in cultured adipose derived stem cells, in a wide perspective. Furthermore, the coalition of cell adhesion molecules with the proliferation potency of MSC and analysis of growth curve of ADSC was also paid accolade. The presence of robust MSC with immense differentiation and transdifferentiation potency was endorsed by lucrative differentiation of P3 cells into mesodermal and neuronal lineages. Additionally, mesenchymal stem cells exhibiting coherent expression of surface markers at P3 in all samples can be cryopreserved for therapeutic applications.

Suppression of zinc finger protein 467 alleviates osteoporosis through promoting differentiation of adipose derived stem cells to osteoblasts

Osteoblast and adipocyte are derived from common mesenchymal progenitor cells. The bone loss of osteoporosis is associated with altered progenitor differentiation from an osteoblastic to an adipocytic lineage. In this study, a comparative analysis of gene expression profiling using cDNA microarray and realtime-PCR indicated that Zinc finger protein 467 (Zfp467) involved in adipocyte and osteoblast differentiation of cultured adipose derived stem cells (ADSCs). Our results showed that RNA interference for Zfp467 in ADSCs inhibited adipocyte formation and stimulated osteoblast commitment. The mRNA levels of osteogenic and adipogenic markers in ADSCs were regulated by si-Zfp467. Zfp467 RNAi in ADSCs could restore bone function and structure in an ovariectomized (OVX)-induced osteoporotic mouse model. Thus Zfp467 play an important role in ADSCs differentiation to adipocyte and osteoblast. This has relevance to therapeutic interventions in osteoporosis, including si-Zfp467-based therapies currently available, and may be of relevance for the use of adipose-derived stem cells for tissue engineering.

Effect of bone defect on expression of molecules related to bone morphogenetic protein signaling pathway in adipose-derived stem cells

ObjectiveTo investigate the molecules related to bone morphogenetic protein (BMP)signaling pathway in adipose-derived stem cells (ADSCs) and the effects of bone defect on their expression in rat endogenous ADSCs.MethodsThirty Wistar rats were randomly divided into a control group (group A, n =5) and an experimental group (group B, n =25). Rats in group A were not treated before their inguinal adipose pads were used to culture ADSCs. Rats in group B were first made into models of bone defect before their inguinal adipose pads were obtained to culture ADSCs in vitro 1, 3, 7, 14 and 21 days after modeling (5 rats for each time). After 7 days of primary culture, the total RNA was extracted from ADSCs to detect the expressions of BMP receptors and Smads by RT-qPCR.ResultsThe expression of BMPR1awas significantly increased in group B at all time points compared with group A ( P ＜ 0. 05 ), and reached the peak at 7 days after bone defect (2. 532,2. 552). The expression of BMPR1b was significantly increased in group B at 1, 3, 7 and 14 days compared with group A ( P ＜ 0. 05), and reached the peak at 14 days after bone defect (6. 628,6. 648). The expressions of BMPR2, Smad5 and Smad8 were significantly increased in group B at 3, 7, 14 and 21 days compared with group A ( P ＜ 0. 05). The expressions of BMPR2 and Smad5 reached the peak at 14 days after bone defect(3. 538, 3. 658; 8. 055, 8. 149), and the expression of Smad8 reached the peak at 7 days (3. 657,3. 759). The expression of Smad1 was significantly increased in group B at 7, 14 and 21 days compared with group A ( P ＜ 0. 05), and reached the peak at 3 days after bone defect (3. 641,3. 771 ).ConclusionsSince ADSCs can express BMP receptors, there may be a BMP signaling pathway in them. A bone defect may possibly induce an increase in expression of the molecules related to BMP signaling pathway in ADSCs. Key words: Adult stem cells; Adipose tissue; Bone morphogenetic protein receptors; Bone defect