Characterization of adipose tissue macrophages and adipose-derived stem cells in critical wounds

Background Subcutaneous adipose tissue is a rich source of adipose tissue macrophages and adipose-derived stem cells which both play a key role in wound repair. While macrophages can be divided into the classically-activated M1 and the alternatively-activated M2 phenotype, ASCs are characterized by the expression of specific stem cell markers. Methods In the present study, we have investigated the expression of common macrophage polarization and stem cell markers in acutely inflamed adipose tissue. Subcutaneous adipose tissue adjacent to acutely inflamed wounds of 20 patients and 20 healthy subjects were harvested and underwent qPCR and flow cytometry analysis. Results Expression levels of the M1-specific markers CD80, iNOS, and IL-1b were significantly elevated in inflammatory adipose tissue when compared to healthy adipose tissue, whereas the M2-specific markers CD163 and TGF-β were decreased. By flow cytometry, a significant shift of adipose tissue macrophage populations towards the M1 phenotype was confirmed. Furthermore, a decrease in the mesenchymal stem cell markers CD29, CD34, and CD105 was observed whereas CD73 and CD90 remained unchanged. Discussion This is the first report describing the predominance of M1 adipose tissue macrophages and the reduction of stem cell marker expression in acutely inflamed, non-healing wounds.

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NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells (ASCs)

10.20944/preprints201701.0088.v1 ◽

2017 ◽

Author(s):

Maria Pitrone ◽

Giuseppe Pizzolanti ◽

Laura Tomasello ◽

Antonina Coppola ◽

Lorenzo Morini ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Embryonic Stem ◽

Stem Cell Markers ◽

Transcription Network ◽

Cell Markers ◽

Nanog Gene ◽

Subcutaneous Adipose

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self- renewal potential and differentiation capacity. Our aim was to study the different expression of embryonic stem cell markers NANOG, SOX2 and OCT3/4 and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting patients (23 men, 47 women; age 45 ± 10; BMI between 25 and 30 range) undergoing elective open-abdominal surgery. Sphere-forming capability was evaluated by plating cells in low adhesion plastic. Stem cell markers CD90 and CD105 were analyzed by flow cytometry and stem cell transcription factors NANOG, SOX2 and OCT3/4 were detected by immunoblotting and Real-Time PCR. NANOG, SOX2 and OCT3/4 interplay was explored by gene silencing. ASCs from VAT and SAT confirmed their mesenchymal stem cell (MSC) phenotype expressing the specific MSC markers CD90 and CD105 and NANOG, SOX2 and OCT3/4. NANOG silencing induced a significant OCT 3/4 (70% ± 0.05) and SOX2 (75% ± 0.03) down-regulation whereas SOX2 silencing did not affect NANOG gene expression. Adipose tissue is an important source of MSC, and siRNA experiments endorse a hierarchical role of NANOG in the complex transcription network that regulates pluripotency and plasticity.

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NANOG Plays a Hierarchical Role in the Transcription Network Regulating the Pluripotency and Plasticity of Adipose Tissue-Derived Stem Cells (ASCs)

10.20944/preprints201701.0088.v2 ◽

2017 ◽

Author(s):

Maria Pitrone ◽

Giuseppe Pizzolanti ◽

Laura Tomasello ◽

Antonina Coppola ◽

Lorenzo Morini ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Embryonic Stem ◽

Stem Cell Markers ◽

Transcription Network ◽

Cell Markers ◽

Nanog Gene ◽

Subcutaneous Adipose

The stromal vascular cell fraction (SVF) of visceral and subcutaneous adipose tissue (VAT and SAT) has increasingly come into focus in stem cell research, since these compartments represent a rich source of multipotent adipose-derived stem cells (ASCs). ASCs exhibit a self- renewal potential and differentiation capacity. Our aim was to study the different expression of embryonic stem cell markers NANOG, SOX2 and OCT3/4 and to evaluate if there exists a hierarchal role in this network in ASCs derived from both SAT and VAT. ASCs were isolated from SAT and VAT biopsies of 72 consenting patients (23 men, 47 women; age 45 ± 10; BMI between 25 and 30 range) undergoing elective open-abdominal surgery. Sphere-forming capability was evaluated by plating cells in low adhesion plastic. Stem cell markers CD90 and CD105 were analyzed by flow cytometry and stem cell transcription factors NANOG, SOX2 and OCT3/4 were detected by immunoblotting and Real-Time PCR. NANOG, SOX2 and OCT3/4 interplay was explored by gene silencing. ASCs from VAT and SAT confirmed their mesenchymal stem cell (MSC) phenotype expressing the specific MSC markers CD90, CD105, NANOG, SOX2 and OCT3/4. NANOG silencing induced a significant OCT 3/4 (70% ± 0.05) and SOX2 (75% ± 0.03) down-regulation whereas SOX2 silencing did not affect NANOG gene expression. Adipose tissue is an important source of MSC, and siRNA experiments endorse a hierarchical role of NANOG in the complex transcription network that regulates pluripotency and plasticity.

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200 TRANSPLANTATION AND IN VIVO DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN SWINE

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab200 ◽

2006 ◽

Vol 18 (2) ◽

pp. 208 ◽

Cited By ~ 1

Author(s):

A. S. Lima ◽

S. A. Malusky ◽

M. R. B. Mello ◽

S. J. Lane ◽

J. R. Rivera ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Cellular Response ◽

Agricultural Research ◽

Primary Concern ◽

Adipose Derived Stem Cells ◽

Subcutaneous Adipose

A primary concern in stem cell biology is that observations made in vitro may be an artifact of the in vitro culture environment. In vitro derived stem cells can be implanted into the environment from which they are derived so that their response to physiological conditions may be observed. Several important cellular characteristics need to be examined following the cell's reintroduction to the in vivo environment, including the potential for differentiation, proliferative ability, and life span. Studying implanted stem cells will assist in determining the potential for stem cell use in clinical therapies and provide further understanding of the role adult stem cells have in the adult body. Currently, the scientific literature is lacking a detailed description of the cellular response of adipose-derived stem cells (ADSCs) reintroduced to their exact tissue of origin. Thus, the aim of this study was to evaluate porcine ADSC growth in vivo and to analyze cell differentiation in vivo following injection of undifferentiated ADSCs into subcutaneous fat. Subcutaneous adipose tissue was isolated from the back fat of male pigs (11 months of age) and digested with 0.075% collagenase at 37�C for 90 min. The digested tissue was centrifuged at 200g for 10 min to obtain a cell pellet. The pellet was re-suspended with DMEM and the ADSCs were plated onto 75 cm2 flasks (5000-10 000 cells per cm2) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% gentamicin. Passage 3 ADSCs were labeled with fluorescent dye (PKH26; Sigma, St. Louis, MO, USA) and sorted by flow cytometry. After sorting, positive cells were washed and re-suspended in culture medium. For transplantation, 100 �L of cell suspension in DMEM containing one of four cell concentrations (0 (control); 30 000; 300 000; and 900 000 cells) were placed in a 1-mL syringe and injected into the subcutaneous back fat of recipient pigs (n = 2). Each pig had previously been tattooed with 12 13 � 13 squares to mark injection sites. The treatments were replicated three times within each animal. Two and three weeks after transplantation, animals were euthanized, the back fat containing the transplantation site was harvested, and the cells were disaggregated as described above. The buoyant adipocytes and pelleted ADSCs cells were then analyzed by flow cytometry. The results indicated that there were dose- and time-dependent increases in labeled ADSCs and labeled adipocytes in the fat samples with increasing cell number (from 0 to 300 000 cells). There was, however, a decrease in labeled ADSCs at the 900 000-cell dose, which is likely due to excess cells being transplanted or an immune reaction. Both of these aspects are currently being evaluated. In conclusion, undifferentiated ADSCs from swine can be isolated from and returned to the subcutaneous adipose layer and differentiate into mature adipocytes. This work was supported by the Council for Food and Agricultural Research (C-FAR) Sentinel Program, University of Illinois.

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Are Adipose-Derived Stem Cells from Liver Falciform Ligaments Another Possible Source of Mesenchymal Stem Cells?

Cell Transplantation ◽

10.3727/096368916x693833 ◽

2017 ◽

Vol 26 (5) ◽

pp. 855-866 ◽

Cited By ~ 4

Author(s):

Sang Woo Lee ◽

Jae Uk Chong ◽

Seon Ok Min ◽

Seon Young Bak ◽

Kyung Sik Kim

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipose Tissue ◽

Liver Disease ◽

Subcutaneous Adipose Tissue ◽

Falciform Ligament ◽

Pcr Analysis ◽

Marker Genes ◽

Adipose Derived Stem Cells ◽

Subcutaneous Adipose

Falciform ligaments in the liver are surrounded by adipose tissue. We investigated the capability of adipose-derived stem cells from human liver falciform ligaments (hLF-ADSCs) to differentiate into hepatic-type cells and confirmed the functional capacity of the cells. Mesenchymal stem cells (MSCs) were isolated from the liver falciform ligament and abdominal subcutaneous adipose tissue in patients undergoing partial hepatectomy for liver disease. Cells were cultivated in MSC culture medium. Properties of MSCs were confirmed by flow cytometry, RT-PCR analysis, immunocytochemistry assays, and multilineage differentiation. Hepatic induction was performed using a three-step differentiation protocol with various growth factors. Morphology, capacity for expansion, and characteristics were similar between hLF-ADSCs and adipose-derived stem cells from human abdominal subcutaneous adipose tissue (hAS-ADSCs). However, hematopoietic– and mesenchymal–epithelial transition (MET)-related surface markers (CD133, CD34, CD45, and E-cadherin) had a higher expression in hLF-ADSCs. The hepatic induction marker genes had a higher expression in hLF-ADSCs on days 7 and 10 after the hepatic induction. Albumin secretion was similar between hLF-ADSCs and hAS-ADSCs at 20 days after the hepatic induction. The hLF-ADSCs had a different pattern of surface marker expression relative to hAS-ADSCs. However, proliferation, multilineage capacity, and hepatic induction were similar between the cell types. Accordingly, it may be a useful source of MSCs for patients with liver disease.

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401 MIGRATION AND THERAPEUTIC POTENTIAL OF PORCINE ADULT ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab401 ◽

2010 ◽

Vol 22 (1) ◽

pp. 357 ◽

Cited By ~ 2

Author(s):

S. M. Wilson ◽

E. Monaco ◽

M. S. Goldwasser ◽

S. G. Clark ◽

W. L. Hurley ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Subcutaneous Adipose Tissue ◽

Adult Stem Cells ◽

Bone Defects ◽

Adipose Derived Stem Cells ◽

Subcutaneous Adipose ◽

Time Point

Bone marrow is one current source of adult stem cells for therapeutic purposes; however, the magnitude and accessibility of subcutaneous adipose tissue in humans make it an attractive alternative. Numerous in vitro studies have been conducted to determine how these cells act in vitro, but it is imperative to determine the vast abilities of these cells in vivo. The objective of this study was to evaluate in vivo migration and bone healing ability after transplanting adipose-derived stem cells (ADSC) in a swine model. Adipose-derived stem cells were isolated from subcutaneous adipose tissue of adult Yorkshire pigs and cultured in vitro. At 80 to 90% confluence/passage 3, the cells were trypsinized and labeled in suspension with carboxyfluorescein succinimidyl ester (CFDA-SE). This project included 20 pigs weighing between 63.5 and 81.7 kg. Bilateral mandibular osteoectomies with 10-mm defects were performed on each pig. Of the 20 pigs, half received a treatment of 2.5 million CFDA-SE labeled stem cells administered directly into each defect (DI), and the remaining half received a treatment of approximately 5 million CFDA-SE labeled stem cells through an ear vein injection via catheter (EVI). The time points were 1 h and 2 and 4 wk, with 2 pigs per time with the DI and EVI treatments. Pigs were slaughtered at each time, and spleen, liver, lung, kidney, ear vein, blood, and mandible tissues were collected. Blood samples were collected from the jugular vein with EDTA and processed via flow cytometry after collection. Tissues were fixed in 10% buffered formalin for histology. Fluorescent microscopy (CFDA-SE excitation/emission is 492/517 nm) has confirmed that transplanted ADSC do indeed migrate to a site of injury or trauma. Labeled cells were also present in blood collected from the 1-h time point group. Currently, we have not seen the presence of labeled ADSC in the other tissues (spleen, liver, lung, and kidney) after the 1-h time point. We did observe that ADSC administered by DI and EVI were able to significantly heal and regenerate bone defects within 4 wk post-surgery (P < 0.05, ANOVA with F-test), in contrast to bone defects in pigs that did not receive cell injections (control). Evidence of ADSC-related healing and bone regeneration was evident by gross visualization, dual-energy x-ray absorptiometry (DXA) and micro computer tomography (microCT) analysis. The clinical implications of these results are significant for treating many diseases in which inflammation or defects exist, such as cardiac disease, neurological disease, or traumatic injuries to both soft and hard tissue. If the adult stem cells can be harvested from fat, encouraged to produce bone or cartilage, and then reinserted into defects, treatment protocols for trauma victims could be developed that would reduce the need for alternate harvesting techniques for bone. This work was support by a grant from the Illinois Regenerative Medicine Institute (IDPH # 63080017).

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Successful Isolation of Viable Adipose-Derived Stem Cells from Human Adipose Tissue Subject to Long-Term Cryopreservation: Positive Implications for Adult Stem Cell-Based Therapeutics in Patients of Advanced Age

Stem Cells International ◽

10.1155/2015/146421 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 14

Author(s):

Sean M. Devitt ◽

Cynthia M. Carter ◽

Raia Dierov ◽

Scott Weiss ◽

Robert P. Gersch ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Cell Isolation ◽

Stem Cell Markers ◽

Adipose Derived Stem Cells ◽

Patient Age ◽

Patient Âgé ◽

Stem Cell Isolation

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days) from patients of varying ages (26–62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved <1 year than from tissue cryopreserved >2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

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