Mesenchymal Stem Cell Markers CD90 Research Articles

Early Results of Male Stress Incontinence Treatment Using Stromal-Vascular Fraction of Autologous Fat Tissue

Introduction. Male stress urinary incontinence (SUI), which affects 10 % of the population, is most commonly caused by the effects of operations on the prostate gland. Despite the presence of a large number of surgical and conservative methods to treat this disease, the problem of choosing treatment tactics is still relevant. There are limited literature data on the effectiveness of using the stromal-vascular fraction (SVF) of autologous fat in the treatment of stress urinary incontinence. The aim of this study is to analyse the usage of SVF in the treatment of SUI patients.Materials and methods. A randomised study was carried out on a group of 8 patients with stress urinary incontinence of mild and moderate severity more than one year after radical prostatectomy or transurethral resection of the prostate. The patients received stromal-vascular fractions of autologous fat into the bladder sphincter area during the period November 2017 — June 2018.Results. After 4 weeks, the Pad Test and ICIQ-SF QoL assessments showed the first positive clinical results and improving quality of life. Checkpoints of 3, 6 and 12 months were selected for measuring results. Cytological analysis of the stromalvascular fraction of autologous fat revealed the expression of mesenchymal stem cell markers CD44, CD73, CD90, which may explain the method’s high efficiency.Conclusion. This work supports the hypothesis that transplantation of SVF from autologous adipose tissue is an effective and safe method for treating patients with incontinence.

Induction of chondrogenic or mesenchymal stem cells from human periodontal ligament cells through inhibition of Twist2 or Klf12.

Periodontitis leads to destruction of periodontal ligament, cementum and alveolar bone. Regeneration of periodontal tissue is dependent on mesenchymal stem cells (MSC) present in the periodontal ligament, and transcription factors determine the direction of MSC differentiation. The present study was conducted to investigate the transcription factors that are crucial for maintaining the characteristics of the periodontal ligament. The mRNA levels of several transcription factors were measured in cultured human periodontal ligament (HPDL) cells, human gingival fibroblasts and osteoblast-like Saos2 cells. HPDL cells were transfected for 72 h with siTwist2, siKlf12, or siMix (siTwist2, siPax9, and siKlf12). The cells were then harvested and subjected to real-time PCR and Western blotting. siTwist2 suppressed the levels of Twist2, Sox2 and Col1a1 mRNAs, and increased those of Sox5 and aggrecan mRNAs. siKlf12 decreased the mRNA levels of Klf12, Runx3, Zfp521, and Stab2, and increased those of Sox2, Klf4, and the MSC markers CD90 and CD105. These results suggest that transfection with siMix and siTwist2 induced chondrogenesis, and that siKlf12 induced the differentiation of MSC in HPDL cells. Thus, inhibition of Twist2 or Klf12 induced the differentiation of chondrogenic or mesenchymal stem cells in this setting, suggesting that the characteristics of HPDL cells may be altered by inhibition of specific transcription factors.

Mesenchymal Stem Cell Marker Expression in Gastric Cancer Stroma.

Spindle-shaped stromal cells of tumors are derived from various cellular origins, including mesenchymal stem cells (MSCs). MSCs express CD73, CD90 and CD105 antigens. Herein, the aim was to investigate the association between the expression of specific MSC markers in gastric cancer stromal cells and the clinicopathological features of the disease. The expression of CD73, CD90 and CD105 in spindle-shaped cancer stromal cells was studied by immunohistochemistry in tissue arrays containing 546 gastric cancer cases. Univariate and multivariate analyses were performed to evaluate the association of MSC marker expression with clinicopathological variables. Spindle-shaped cancer stromal cells expressing the MSC markers CD73, CD90 or CD105 were associated with larger tumor size, advanced cancer, venous infiltration, lymphatic infiltration, and lymph node metastasis. Statistical analysis demonstrated that the presence of CD105-positive spindle cells was an independent prognostic factor of advanced cancer, lymph node metastasis and EBV infection in multivariate analysis. Spindle-shaped gastric cancer stromal cells expressing CD73, CD90 or CD105 are involved in disease progression, and among them, CD105-positive cells are strongly associated with poor prognosis.

Human amniotic membrane as differentiating matrix for in vitro chondrogenesis.

The aim of the present study is to use human amniotic membrane (HAM) for in vitro chondrogenesis of placenta-derived mesenchymal stem cells (MSCs) and umbilical cord-derived MSCs. MSCs from the placenta and umbilical cord were isolated, characterized by immunophenotyping and after analyzing their rate of proliferation, cytotoxicity and viability, chondrogenesis was performed on plastic adherent surface and on HAM. Successfully isolated and characterized placenta-derived MSCs and umbilical cord-derived MSCs revealed positive expression of MSCs markers CD90, CD73, CD105 and CD49d, while they were negative for CD45. Both types of cells in the presence of chondrogenic induction medium on plastic adherent surface and HAM showed aggregates of proteoglycan and strong expression of COL2A1 (collagen 2) and ACAN1 (aggrecan). HAM supported proliferation as well as chondrogenesis of MSCs and provide novelty of HAM utilization as an efficient natural delivery matrix for stem cell transplantation.

Generation of Mesenchymal Stem Cells from Human Embryonic Stem Cells in a Complete Serum-free Condition.

Mesenchymal stem cells (MSC) have been derived from a variety of tissues, and cultured either in animal serum-containing (SC) or serum-free (SF) media. We have previously derived MSC from human embryonic stem cells via an intermediate trophoblast step (named EMSC), which also have immunosuppressive and therapeutic effects on animal models of autoimmune disease. To promote the clinical application of this new source of MSC, we report here EMSC derived and cultured in a SF medium MesenCult (SF-EMSC) in comparison with a SC medium (SC-EMSC). SF-EMSC derived in MesenCult also expressed typical MSC markers CD73, CD90, and CD105, and manifested multipotency to differentiate to osteocytes, chondrocytes, and adipocytes. Comparably, CD105+ cells reached 90% about one week slower in the SF than SC conditions, and the proliferation rate was slightly faster for SF-EMSC than SC-EMSC at later passages. Both SF- and SC-EMSC responded similarly to the inflammatory stimulus IFNγ. However, the inflammatory cytokines IL-6 and IL-8 were expressed much less in SF-EMSC than SC-EMSC. Furthermore, knockdown of P16INK4A in both SF- and SC-EMSC reduced replicative senescence. Together, our results suggest that EMSC can be generated in a complete SF condition, and SF-EMSC are largely similar to SC-EMSC. However, it takes longer time to derive EMSC in the SF than SC conditions, and the SF-EMSC proliferate faster at later passages and produce less of the inflammatory cytokines IL-6 and IL-8 than SC-EMSC. This study provides important information for production of clinically applicable EMSC.

Identification of perivascular and stromal mesenchymal stem/progenitor cells in porcine endometrium.

Mammalian uterus contains a population of mesenchymal stem/progenitor cells that likely contribute to endometrial regeneration during each reproductive cycle. In human and mouse, they reside in perivascular, epithelial and stromal compartments of the endometrial functionalis and basalis. Here, we aimed to identify tissue resident cells expressing mesenchymal stem cell markers CD29, CD44, CD90, CD105, CD140b and CD146 in the porcine endometrium. We used single immunofluorescence and Western blotting. Each of these markers was detected in small cells surrounding endometrial blood vessels. CD105 and CD146 were also expressed in single stromal cells. A few stromal and perivascular cells showed the presence of pluripotency marker Oct4 in the cytoplasm, but not in the nucleus, which may imply they are not truly pluripotent. Endometrial cell cultures were examined for the expression of CD29, CD44, CD90, CD105 and CD140b proteins and tested in wound-healing assay and culture model of chemotaxis. In conclusion, our results demonstrate perivascular location of prospective mesenchymal stem/progenitor cells in the porcine endometrium and may suggest that stromal CD105+ and CD146+ cells represent more mature precursors originating from their perivascular ancestors.

ISOLAMENTO E CULTIVO DE CÉLULAS TRONCO MESENQUIMAIS EXTRAÍDAS DO TECIDO ADIPOSO E DA MEDULA ÓSSEA DE CÃES

Resumo Objetivou-se estabelecer um protocolo para extração, cultivo e expansão de células tronco mesenquimais (CTM), utilizando-se 3,0 mL da medula óssea e 3,0 cm3 de tecido adiposo do subcutâneo de três cães machos com seis meses de idade. As amostras foram processadas e as células extraídas e cultivadas em DMEM. Para comprovação do isolamento de CTM, procedeu-se a caracterização fenotípica e a diferenciação osteogênica, adipogênica e condrogênica. As células isoladas apresentaram morfologia alongada e fusiforme e capacidade de se diferenciar em osteoblastos, adipócitos e condrócitos. A caracterização fenotípica revelou alta expressão de marcadores de CTM CD90 (80,04%) e CD29 (96%) nas células de origem medular e CD90 (60,94%) e CD29 (77,08%) nas de origem adiposa. A expressão de marcadores hematopoiéticos foi baixa tanto nas células de origem medular CD45 (1,45%) e CD34 (1,53%), quanto nas de origem adiposa CD45 (1,45%) e CD34 (1,53%). As modificações e adaptações realizadas nos protocolos clássicos simplificaram o processo e foram eficientes, permitindo o isolamento e cultivo de CTM da medula óssea e do tecido adiposo de cães.

Isolation and in vitro cultivation of human urine-derived cells: an alternative stem cell source.

For in vitro tissue engineering in urology, stem cells are commonly isolated from tissue specimens obtained during open or endoscopic surgery. Within the context of the present study our aim was to isolate cells from human urine by an alternative and safe technique rather than using the indicated method. After human urine samples had been collected from young and healthy donors via urethral catheterization, cells were precipitated by centrifugation and cultured. Following this isolation procedure, cells were characterized by immunocytochemical method using specific antibodies. When these cells were characterized by immunocytochemical methods using specific antibodies some of them were positive for mesenchymal stem cell marker CD90 while the others were labelled with urothelial marker cytokeratin 7. When all these results were taken into consideration, urothelial cells together with stem cells were observed in the urine- derived cell population. According to the results obtained from this study human urine may be preferred as an alternative stem cell and urothelial cell source in that this method is and easily reproducible non-invasive method.

SET/I2PP2A overexpression induces phenotypic, molecular, and metabolic alterations in an oral keratinocyte cell line.

The multifunctional SET/I2PP2A protein is known to be overexpressed in head and neck squamous cell carcinoma. However, SET has been reported to have apparently conflicting roles in promoting cancer cell survival under oxidative stress conditions and preventing invasion and metastasis, complicating efforts to understand the contribution of SET to carcinogenesis. In the present study, we overexpressed SETin a spontaneously immortalized oral keratinocyte cell line (NOK-SI SET) and demonstrated that SET upregulation alone was sufficient to transform cells. In comparison with NOK-SI cells, NOK-SI SET cells demonstrated increased levels of phosphorylated Akt, c-Myc and inactive/phosphorylated Rb, together with decreased total Rb protein levels. In addition, NOK-SI SET cells presented the following: (a) a spindle-cell shape morphology compared with the polygonal morphology of NOK-SI cells; (b) loss of mesenchymal stem cell markers CD44 and CD73, and epithelial cell markers CD71 and integrin α6/β4; (c) the ability to form xenograft tumors in nude mice; and (d) increased mitochondrial respiration accompanied by decreased ROSlevels. Overall, our results show that SEToverexpression promotes morphological and oncogenic cell transformation of an oral keratinocyte cell.

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

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 the embryonic stem cell markers NANOG (homeobox protein NANOG), SOX2 (SRY (sex determining region Y)-box 2) and OCT4 (octamer-binding transcription factor 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 ± 5 and 30 ± 5 range) undergoing elective open-abdominal surgery. Sphere-forming capability was evaluated by plating cells in low adhesion plastic. Stem cell markers CD90, CD105, CD29, CD31, CD45 and CD146 were analyzed by flow cytometry, and the stem cell transcription factors NANOG, SOX2 and OCT4 were detected by immunoblotting and real-time PCR. NANOG, SOX2 and OCT4 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 OCT4. NANOG silencing induced a significant OCT4 (70 ± 0.05%) and SOX2 (75 ± 0.03%) downregulation, 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.

Diabetes Impairs the Ability of Skeletal Muscle Pericytes to Augment Postischemic Neovascularization in Mice

Peripheral artery disease is an atherosclerotic disease that causes limb ischemia and has few effective treatments. Stem cell therapy is a promising treatment option, but concomitant diabetes may limit its effectiveness. PURPOSE: To evaluate the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization following the induction of limb ischemia in wild type (WT) and type 2 diabetic (T2DM) mice. METHODS: Pericytes were isolated via fluorescence activated cell sorting for CD45-CD34-CD146+ cells, and pericyte phenotype was confirmed via surface marker expression, gene expression, and in vitro differentiation potential. WT C57BL/6 (n=10) and db/db T2DM (n=8) mice underwent unilateral femoral artery ligation to induce limb ischemia. 24 hrs post-ligation, pericytes or vehicle control were transplanted into the muscles of the ischemic hindlimbs. Postischemic neovascularization was assessed via foot blood flow at pre-surgery, post-surgery, and postoperative days 3, 7, 14, 21, and 28 using laser Doppler perfusion imaging. Differences in gene expression were determined using t-tests; differences in blood flow were determined using linear mixed models. RESULTS: CD45-CD34-CD146+ pericytes were positive for mesenchymal stem cell markers CD90 (74%) and CD105 (65%) and weakly positive for the pericyte marker PDGFRβ (42%) and the endothelial cell marker CD144 (36%). Pericytes transdifferentiated into skeletal myocytes, adipocytes, osteocytes, and endothelial cells. Pericytes had significantly (p<0.05) upregulated Sca1 (4.0-fold) gene expression, downregulated CD31 (0.2-fold) gene expression, and no difference in MyoD (1.0-fold), Pax3 (1.3-fold), or Pax7 (1.0-fold) expression. Blood flow recovery in WT mice was significantly higher after pericyte transplantation than after vehicle control (p=0.03; 81.1±6% vs. 64.7±9% at postoperative day 28). Blood flow recovery in T2DM mice after pericyte transplantation was not different than after vehicle control (p=0.44; 50.1±4% vs. 53.5±4% at postoperative day 28). CONCLUSION: T2DM negates the capacity of pericytes to augment neovascularization after limb ischemia. Further study is required to determine the mechanism by which T2DM impairs this pericyte function. Support: AAUW American Dissertation Fellowship

Survival of the Apical Papilla and Its Resident Stem Cells in a Case of Advanced Pulpal Necrosis and Apical Periodontitis

IntroductionApical papilla represents a source of an enriched mesenchymal stem cell (MSC) population (stem cells of the apical papilla [SCAPs]) that modulates root development and may participate in regenerative endodontic procedures in immature teeth with pulp necrosis. The characteristics and phenotype of this tissue in the presence of inflammation are largely unknown. The purpose of this study was to characterize a human apical papilla sample that was isolated from an immature tooth with pulp necrosis and apical periodontitis. MethodsInflamed periapical tissue that included part of the apical papilla (apical papilla clinical sample [CS]) was collected from an immature mandibular premolar previously diagnosed with pulp necrosis and apical periodontitis during an apexification procedure. Harvested cells from this tissue (SCAP CS) were compared with inflamed periapical progenitor cells (IPAPCs) and normal SCAP (SCAP-RP89) in flow cytometry and quantitative osteogenesis experiments. Part of the issue was further processed for immunohistochemistry and compared with apical papilla and coronal pulp sections from normal immature teeth as well as inflamed periapical tissues from mature teeth. ResultsSimilar to SCAP-RP89, 96.6% of the SCAP CS coexpressed the MSC markers CD73, CD90, and CD105, whereas only 66.3% of IPAPCs coexpressed all markers. The SCAP CS showed a significantly greater mineralization potential than both SCAP-RP89 and IPAPCs. Finally, immunohistochemical analysis revealed moderate infiltration of cells expressing the inflammatory markers CD45/68 in the apical papilla CS and prominent CD24, CD105, and von Willebrand factor expression. ConclusionsUnder inflammatory conditions, human apical papilla was found moderately inflamed with retained SCAP vitality and stemness and increased osteogenic and angiogenesis potential.

Identification and characterization of adult mouse meniscus stem/progenitor cells

ABSTRACTMeniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

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

BackgroundSubcutaneous 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.MethodsIn 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.ResultsExpression 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.DiscussionThis 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.

Differentiation of mesenchymal stem cells from human amniotic fluid to vascular endothelial cells

Endothelial dysfunction is a principle feature of vascular-related disease. Endothelial cells have been acquired for the purposes of the restoration of damaged tissue in therapeutic angiogenesis. However, their use is limited by expansion capacity and the small amount of cells that are obtained. Human amniotic fluid mesenchymal stem cells (hAF-MSCs) are considered an important source for vascular tissue engineering. In this study, hAF-MSCs were characterized and then induced in order to differentiate into the endothelial-like cells. Human amniotic fluid cells (hAFCs) were obtained from amniocentesis at the second trimester of gestation. The cells were characterized as mesenchymal stem cells by flow cytometry. The results showed that the cells were positive for mesenchymal stem cell markers CD44, CD73, CD90 and HLA-ABC, and negative for CD31, Amniotic fluid stem cells marker: CD117, anti-human fibroblasts, HLA-DR and hematopoietic differentiation markers CD34 and CD45. The hAF-MSCs were differentiated into endothelial cells under the induction of vascular endothelial growth factor (VEGF) and analyzed for the expression of the endothelial-specific markers and function. The expression of the endothelial-specific markers was determined by reverse transcriptase-quantitative PCR (RT-qPCR), while immunofluorescent analysis demonstrated that the induced hAF-MSCs expressed von Willebrand factor (vWF), vascular endothelial growth factor receptor 2 (VEGFR2), CD31 and endothelial nitric oxide synthase (eNOS). The network formation assay showed that the induced hAF-MSCs formed partial networks. All results indicated that hAF-MSCs have the potential to be differentiated into endothelial-like cells, while human amniotic fluid might be a suitable source of MSCs for vascularized tissue engineering.

Compatibility of Porous Chitosan Scaffold with the Attachment and Proliferation of human Adipose-Derived Stem Cells In Vitro

Adipose-derived stem cells (ASCs) have potential applications in the repair and regeneration of various tissues and organs. The use of various scaffold materials as an excellent template for mimicking the extracellular matrix to induce the attachment and proliferation of different cell types has always been of interest in the field of tissue engineering because ideal biomaterials are in great demand. Chitosan, a marine polysaccharide, have wide clinical applications and it acts as a promising scaffold for cell migration and proliferation. ASCs, with their multi-differentiation potential, and chitosan, with its great biocompatibility with ASCs, were investigated in the present study. ASCs were isolated and were characterized by two different methods: immunocytochemistry and flow cytometry, using the mesenchymal stem cell markers CD90, CD105, CD73 and CD29. The ASCs were then induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. These ASCs were incorporated into a porous chitosan scaffold (PCS), and their structural morphology was studied using a scanning electron microscope and hematoxylin and eosin staining. The proliferation rate of the ASCs on the PCS was assessed using a PrestoBlue viability assay. The results indicated that the PCS provides an excellent template for the adhesion and proliferation of ASCs. Thus, this study revealed that PCS is a promising biomaterial for inducing the proliferation of ASCs, which could lead to successful tissue reconstruction in the field of tissue engineering.

Isolation, characterization and multi-lineage differentiation of stem cells from human exfoliated deciduous teeth.

The aim of the present study was to isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential. Three SHED cell strains were successfully isolated from three exfoliated deciduous teeth from different human subjects using the outgrowth method. Flow cytometric analysis indicated that SHEDs displayed high expression of the mesenchymal cell markers CD73 and CD90 but low expression of the hematopoietic stem cell marker CD34. PCR analysis illustrated that SHEDs expressed the mesenchymal stem cell markers CD44, CD73 and CD90, the osteoblast markers Alpl, Runx2, CBFA1 and collagen I, the cartilage cell markers Col10a1 and Acan, the adipose cell markers PPARγ2 and LPL, and the neuronal stem cell marker Nestin. In vitro induction experiments demonstrated the potential of the SHEDs for osteogenic, adipogenic and neurogenic differentiation. These SHED cells may be useful for further stem cell research and future therapeutic applications.

Isolation and Characterization of Pericytes from Murine Skeletal Muscle

Pericytes are multipotent stem cells located in the perivascular space throughout the microvasculature. Their ability to differentiate into mesodermal cell types and modulate the local tissue environment through paracrine signaling makes pericytes a novel therapeutic target, but their prospective isolation and identification preclude their widespread use in therapeutic research. PURPOSE: To use novel flow cytometry methods to isolate and characterize pericytes from murine skeletal muscle. METHODS: Gastrocnemius and soleus complexes were dissected from CD1 mice, aged 3 months. Muscles were digested using collagenase into a single cell suspension. Cells were filtered through 70 um and 40 um mesh then resuspended in staining medium. Cells were stained at 4°C for 20 minutes with the following antibodies: CD45-BB515 (1:400), CD34-BV421 (1:100), CD56-PE (1:100) and CD146-Alexa647 (1:100). Fluorescence activated cell sorting (FACS) was used to sort CD45-CD56-CD34-CD146+ pericytes using appropriate unstained, single stained, and fluorescence minus one controls. P0 cells were plated on 0.2% gelatin coated plates in EBM-2 complete medium. At 70% confluence, P0 cells were split 1:1 on polystyrene tissue culture plates, and split 1:2 thereafter. Flow cytometry was used to determine the expression of pericyte markers (NG2 and PDGFβ), mesenchymal cell markers (CD73, CD90, and CD105), endothelial (CD31) and hematopoietic cells (CD45) markers on P5 cells. Characterization data were analyzed for the percentage of positive cells and median fluorescent intensity relative to unstained control (nMFI). RESULTS: Pericytes were positive for the pericyte marker CD146 (15.5%, nMFI 102.4), weakly positive for PDGFRβ (10.8%, nMFI 7.5), and weakly positive for NG2 (7.0%, nMFI 6.9). Cultured pericytes were positive for mesenchymal stem cell markers CD73 (52.9%, nMFI 56.9), and CD105 (35.3%, nMFI 9.5), but negative for CD90 (4.9%, nMFI 6.3). Pericytes were negative for the endothelial marker CD31 (2.1%, nMFI 4.4) and hematopoietic marker CD45 (5.4%, nMFI 7.9). CONCLUSIONS: FACS sorting for CD45-CD56-CD34-CD146+ cells yields murine pericytes that can be reliably isolated, definitively characterized, and may be suitable for in vivo and in vitro experiments to test the efficacy of pericyte cell therapy for various diseases.

Isolation and Characterization of Multipotent Precursor Cells from Murine Adipose Tissue using a Clinically Approved Cell Separation System

Recent studies underscored the clinical potential of adipose-derived multipotent stem-/precursor cells (ASPCs). One of the main hurdles en route to clinical application was to isolate cells without having to perform expansion cultures outside the OR. A new generation of clinically approved, commercially available cell separation systems claims to provide ASPCs ready for application without further expansion cultures. However, it is unclear if the new systems yield sufficient cells of adequate quality for the use in autologous murine models. The aim of this study was to isolate and characterize adipose-derived precursor cells taken from the inguinal fat pat of wistar rats using InGeneron's clinically approved ARC™-cell separation system. We isolated cells from the inguinal fat pad of 3 male Wistar rats according to the manufacturer's protocol. In order to reduce the influence of the atmospheric oxygen on the multipotent precursor cells, one half of the cell suspension was cultivated under hypoxia (2% O2) simulating physiological conditions for ASPCs. As a control, the other half of the cells were cultivated under normoxia (21% O2). Cell surface markers CD90, CD29, CD45 and CD11b/c were analyzed by FACS, and osteogenic and adipogenic differentiation of the ASPCs was performed. Finally, cellular growth characteristics were assessed by evaluation of the cumulative population doublings and CFU assay, and metabolic activity was evaluated by WST-1 assay. Processing time was 90 (± 12) min. 1 g of adipose tissue yielded approximately 60 000 plastic adhering cells. Both groups showed a high expression of the mesenchymal stem cell markers CD90 and CD29 while they were negative for the leucocyte markers CD45 and CD11b/c. A strong osteogenic differentiation and a sufficient adipogenic differentiation potential was proven for all ASPCs. Under hypoxia, ASPCs showed increased proliferation characteristics and CFU efficiency as well as a significantly increased metabolic activity. This study showed that sufficient multipotent ASPCs of appropriate quality can be isolated from the inguinal fat pad of Wistar rats using the ARC™-cell separation system. As shown in previous studies, cultivation of cells under hypoxic conditions increased their stemness. Our findings will enable future studies that focus on autologous transplantation of ASPCs in a rat model, which most closely resembles a possible clinical application.

Human Chorionic Villus Stem Cells May Contribute to Endogenous Factor VIII Production during Gestation

INTRODUCTION: The placenta is an organ that plays a vital role in providing nutrition, elimination of metabolic products and gas exchange for the growing fetus. Major organs that play these roles in an adult are undergoing development during gestation. The liver, a metabolic organ with a variety of endocrine and exocrine functions including the synthesis of coagulation proteins, is the major site of hematopoiesis at 11-15 weeks during gestation. Production of coagulation proteins during gestation remains unclear. We investigated the potential of very early gestation human chorionic villus mesenchymal stem cells (PMSCs) for Factor VIII (FVIII) mRNA and production of biologically active FVIII.METHODOLOGY: PMSCs were isolated from human placenta at 9-13 weeks of gestational age by tissue explant culture and characterized via flow cytometry for mesenchymal stem cell (MSC) markers. Tri-lineage differentiation of PMSCs was investigated by inducing PMSCs in different culture conditions. Polymerase chain reaction (RT-PCR) was performed on cell lysates using FVIII primers. Chromogenic assay was performed on culture medium of PMSCs and HUVECs (negative control) after 72 hours in culture. Human plasma was used as positive control.RESULTS: PMSCs isolated from preterm human placenta were plastic adherent, showed spindle-shaped morphology and demonstrated expression of MSC markers CD105, CD90, CD73, CD44, and CD29, and did not express CD184, HLA-DR or hematopoietic and endothelial markers CD45, CD34 and CD31. Tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages was observed under different culture conditions. These results show multi-potency and a surface marker profile analogous to bone marrow mesenchymal stem cells (BMSCs). PCR analysis revealed FVIII mRNA expression on PMSCs and was negative on HUVECs. Chromogenic assay showed ~0.4 U/ml on PMSCs and ~0.1 U/ml in HUVECs.CONCLUSION: Here we report that early gestation PMSCs express and secrete biologically active FVIII. Further studies using different gestational ages of the placenta is needed to understand the potential contribution of chorionic villus to FVIII production during gestation. DisclosuresPowell:CSL Behring: Employment.