Nuclear heterogeneity and proliferation activity of human adipose derived MSC-like cells

Adipose tissue (AT) is an easily available source of mesenchymal-like stem cells (MSCs) that are appropriate for applications in regenerative medicine. There is conflicting evidence on the morphology of AT-derived stem cells (ADSCs). Here, we described the morphology and proliferation activity of human ADSCs. The cells were plated at a density of 10 cells/sm² and cultivated for 1 month. Twenty-one colonies were grown. In nine out of 17 analyzed colonies, few atypical cells (large nuclei and cytoplasm) were found. ANOVA demonstrated that colonies also differed (p = 0.0025) in the size and diameter of cell nuclei. The size of nuclei and logarithm of cell density were correlated in the reverse proportion (−0.7; p = 0.002). Thus, a culture obtained from the stromal vascular fraction (SVF) is heterogeneous and composed of two types of cells, i.e., highly proliferative and large, low proliferative cells. These cells are typical of the MSC and ADSC cultures described in literature. The cell heterogeneity observed in some colonies probably resulted from variations in the cell-cycle phases.     

Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.     

Effect of cell senescence on the impedance measurement of adipose tissue-derived stem cells

Label-free and real-time monitoring of stem cells based on electrical impedance measurement is increasingly utilized for the quality control of the isolated stem cells to be used in stem cell-based tissue therapy or regenerative medicine. In spite of that the proliferative capacity and multipotency of stem cells are dependent on the type and age of the source tissue, however, the effect of the cell senescence on the impedance measurement of stem cells has not yet been studied. We investigated whether the senescence of adipose tissue-derived stem cells (ADSCs) can be detected by electrical impedance spectroscopy. For this, ADSCs at passage 9 and 31 were prepared and those genetic characteristics and growth kinetics were evaluated by quantitative polymerase chain reaction and cell counting. While the identified ADSCs were grown on the indium tin oxide electrodes, the impedance spectra were measured and interpreted by fitting analysis with an equivalent circuit model. ADSCs at passage 9 adhered on the electrode were small and spindle-shaped whereas the cells at passage 31 were flattened and larger than younger cells. At the beginning of culture time when the cell adhesion occurred, the resistance at 4.6 kHz of passage 31 cells was higher than passage 9 due to the larger size of older cells. Afterwards, the value of passage 9 cells increased higher than passage 31, since younger cells proliferated more than old cells. Therefore, the impedance measurement could characterize the proliferative capacity of ADSCs during expanded culture.     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

The growth and morphological characteristics of undifferentiated and differentiated cells of the F9 mouse teratocarcinoma line

Differentiation of the F9 cell line was induced by treating the cells with retinoic acid (10(-6) M) and dibutyryl cycloadenosinemonophosphate (10(-4) M). The population doubling time and the portion of cells in G1-phase increase and saturation density falls as the result of this treatment. Differentiated F9 cells demonstrate a decreased capacity of forming colonies in the soft agar, lose their capacity of proliferating at the clonal density, and acquire the limited life-span in culture after reseeding at a high density. Some cells in the differentiated population retain their capacity of forming colonies in the soft agar and (or) of binding antibodies against the stem cell marker SSEA-1. Cells with the stem cell morphology were found in the course of passaging of differentiated cells after reseeding at a high density. These cells were able to differentiate after the standard procedure of the induction of differentiation with retinoic acid and dibutyryl cAMP. Causes of the rising and supporting of heterogeneity of the differentiated F9 cells are discussed.     

Schwann-like cell differentiation of rat adipose-derived stem cells by indirect co-culture with Schwann cells in vitro

Objectives: Schwann cell (SC) transplantation is a promising therapy for peripheral nerve transaction, however, clinical use of SCs is limited due to their very limited availability. Adipose‐derived stem cells (ADSCs) have been identified as an alternative source of adult stem cells in recent years. The aim of this study was to evaluate the feasibility of using ADSCs as a source of stem cells for differentiation into Schwann‐like cells by an indirect co‐culture approach, in vitro. Materials and methods: Multilineage differentiation potential of the obtained ADSCs was assayed by testing their ability to differentiate into osteoblasts and adipocytes. The ADSCs were co‐cultured with SCs to be induced into Schwann‐like cells through proximity, using a Millicell system. Expression of typical SC markers S‐100, GFAP and P75NTR of the treated ADSCs was determined by immunocytochemical staining, western blotting and RT‐PCR. Myelination capacity of the differentiated ADSCs (dADSCs) was evaluated in dADSC/dorsal root ganglia neuron (DRGN) co‐cultures. Results: The treated ADSCs adopted a spindle shaped‐like morphology after co‐cultured with SCs for 6 days. All results of immunocytochemical staining, western blotting and RT‐PCR showed that the treated cells expressed S‐100, GFAP and P75NTR, indications of differentiation. dADSCs could form Schwann‐like cell myelin in co‐culture with DRGNs. Undifferentiated ADSCs (uADSCs) did not form myelin compared to DRGNs cultured alone, but could produce neurite extension. Conclusions: These results demonstrate that this indirect co‐culture microenvironment could induce ADSCs to differentiate into Schwann‐like cells in vitro, which may be beneficial for treatment of peripheral nerve injuries in the near future.     

Effect of Calcitonin Gene-Related Peptide on the Neurogenesis of Rat Adipose-Derived Stem Cells In Vitro

Calcitonin gene-related peptide (CGRP) promotes neuron recruitment and neurogenic activity. However, no evidence suggests that CGRP affects the ability of stem cells to differentiate toward neurogenesis. In this study, we genetically modified rat adipose-derived stem cells (ADSCs) with the CGRP gene (CGRP-ADSCs) and subsequently cultured in complete neural-induced medium. The formation of neurospheres, cellular morphology, and proliferative capacity of ADSCs were observed. In addition, the expression of the anti-apoptotic protein Bcl-2 and special markers of neural cells, such as Nestin, MAP2, RIP and GFAP, were evaluated using Western blot and immunocytochemistry analysis. The CGRP-ADSCs displayed a greater proliferation than un-transduced (ADSCs) and Vector-transduced (Vector-ADSCs) ADSCs (p<0.05), and lower rates of apoptosis, associated with the incremental expression of Bcl-2, were also observed for CGRP-ADSCs. Moreover, upon neural induction, CGRP-ADSCs formed markedly more and larger neurospheres and showed round cell bodies with more branching extensions contacted with neighboring cells widely. Furthermore, the expression levels of Nestin, MAP2, and RIP in CGRP-ADSCs were markedly increased, resulting in higher levels than the other groups (p<0.05); however, GFAP was distinctly undetectable until day 7, when slight GFAP expression was detected among all groups. Wnt signals, primarily Wnt 3a, Wnt 5a and β-catenin, regulate the neural differentiation of ADSCs, and CGRP gene expression apparently depends on canonical Wnt signals to promote the neurogenesis of ADSCs. Consequently, ADSCs genetically modified with CGRP exhibit stronger potential for differentiation and neurogenesis in vitro, potentially reflecting the usefulness of ADSCs as seed cells in therapeutic strategies for spinal cord injury.     

Cell kinetic study on GM colonies using autoradiography and collagen gel culture with special reference to unique proliferation of eosinophils

Using a newly developed technique of autoradiography and collagen gel culture, a kinetic study on human GM colonies was attempted. Colonies of immature cells appeared first on day 5. The number of mixed colonies (mixture of immature cells, neutrophils, and/or monocyte-macrophages) and neutrophil colonies attained a maximum on days 8 to 10 and a broad peak of monocyte-macrophage colonies was observed on days 11 to 16. Eosinophil colonies appeared first on day 12, reached a maximum on day 18, and then gradually decreased. A detailed analysis of the order of appearance of the colonies suggests that mixed, neutrophil and monocyte-macrophage colonies originate from immature cell colonies or clusters, while eosinophil colonies do not. An autoradiographic study was designed to study the proliferation characteristics of each colony. Labeling indices (LI) with 3H-TdR of the cells in immature cell colonies were always high. LI of the cells in differentiated colonies such as neutrophil, monocyte-macrophage, and mixed colonies were low throughout the observation period. In contrast, LI of the cells in eosinophil colonies were constantly high regardless of the size of cell aggregates and the duration of the culture period. Both mitotic indices and mean grain counts on the nuclei of eosinophils were similar to those of immature cells. These results suggest that eosinophil colonies develop from their own small clusters and that eosinophils retain a fairly good proliferative capacity even when differentiated to the level in which specific granules appear in the cytoplasm.     

Factors affecting the generation of mast cells from multipotential colony-forming cells

The cells responsible for the long-term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed-erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM). Mixed-erythroid colonies from 14-day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed-erythroid colonies. Thus, mixed-erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.     

Purification and growth of endothelial progenitor cells from murine bone marrow mononuclear cells

This study reports the culture and purification of murine bone marrow endothelial progenitor cells (EPCs) using endothelial cell-conditioned medium (EC-CM). Endothelial-like cells appeared at day 5 in culture of bone marrow mononuclear cells in the presence of EC-CM in the culture system, and these cells incorporated acetylated low-density lipoproteins (Ac-LDL) and reacted with endothelial-specific Ulex Europaeus Lectin. Continued incubation of these cells at low density with EC-CM for longer than 10 days resulted in the formation of endothelial cell colonies which gave rise to colonies of endothelial progeny and can be passed for many generations in the EC-CM culture system. Cells derived from these colonies expressed endothelial cell markers such as vWF and CD31, incorporated Dil-Ac-LDL, stained positive for Ulex Europaeus Lectin, formed capillary-like structures on Matrigel, and demonstrated a high proliferative capacity in culture. These bone marrow-derived adherent cells were identified as EPCs. The purification and the formation of EPC colonies by using EC-CM were associated with the cytokines secreted in the EC-CM. VEGF, bFGF, and GM-CSF in the EC-CM stimulated the proliferation and growth of EPCs, whereas AcSDKP (tetrapeptide NAc-Ser-Asp-Lys-Pro) in EC-CM suppressed the growth of mesenchymal stem cells (MSC) and fibroblasts. This approach is efficient for isolation/purification and outgrowth of bone marrow EPCs in vitro, a very important cell source in angiogenic therapies and regenerative medicine.     

In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.     

Aldehyde dehydrogenase activity helps identify a subpopulation of murine adipose-derived stem cells with enhanced adipogenic and osteogenic differentiation potential

AIM To identify and characterize functionally distinct subpopulation of adipose-derived stem cells(ADSCs).METHODS ADSCs cultured from mouse subcutaneous adipose tissue were sorted fluorescence-activated cell sorter based on aldehyde dehydrogenase(ALDH) activity, a widely used stem cell marker. Differentiation potentials were analyzed by utilizing immunocytofluorescece and its quantitative analysis.RESULTS Approximately 15% of bulk ADSCs showed high ALDH activity in flow cytometric analysis. Although significant difference was not seen in proliferation capacity, the adipogenic and osteogenic differentiation capacity was higher in ALDHHi subpopulations than in ALDHLo. Gene set enrichment analysis revealed that ribosome-related gene sets were enriched in the ALDHHi subpopulation. CONCLUSION High ALDH activity is a useful marker for identifying functionally different subpopulations in murine ADSCs. Additionally, we suggested the importance of ribosome for differentiation of ADSCs by gene set enrichment analysis.     

Phenotypic heterogeneity within clones of fetal human cells.

The heterogeneity of cell morphology characteristics of some colonies of human fetal kidney and amniotic fluid cells has been analyzed by biochemical and cell-cloning techniques. All the presumed subclones derived from dimorphic colonies were initially epithelioid, but some cells became fibroblastlike as the cell density increased. To determine if the observed heterogeneity occurred within clonal populations of cells, we determined the isozyme phenotype of dimers from renal cells heterozygous for glucose-6-phosphate dehydrogenase (G6PD). Colonies showing mixed cellular morphology expressed only a single G6PD isozyme, thus revealing their single-cell origin. Our results indicate that cell morphology is influenced by the cellular density within the clone, and that a single human renal cell in vitro can yield progeny of two morphological types.     

Heritability of in vitro phenotypes exhibited by murine adipose-derived stromal cells

Adipose-derived stromal cells (ADSCs) exhibit significant potential as therapeutic agents to promote tissue regeneration. Success of ADSC-based therapies is dependent upon efficient cell expansion in vitro as well as postinjection survival in the caustic milieu of damaged tissue. Genetic background regulates ADSC proliferative capacity and stress resistance, but the extent of the genetic effect size is not completely defined. The present study aimed to quantify phenotypic ranges and heritability of in vitro ADSC characteristics. ADSCs were isolated from mice representing 16 genetically diverse inbred mouse strains, including 12 classical inbred strains and four wild-derived strains. Cells were grown in vitro, and proliferative capacity and oxidative stress resistance were assessed. The fold change for ADSC growth ranged from 0.87 (BALB/cByJ) to 23.60 (POHN/DehJ), relative to original seeding density. The heritability of proliferative capacity was estimated to be 0.6462 (p = 9.967 × 10^?15), and this phenotype was not associated with other ADSC traits. Cell viability following H₂O₂ treatment ranged from 39.81 % (CAST/EiJ) to 91.60 % (DBA/2 J), and the heritability of this phenotype was calculated as 0.6146 (p = 1.22 × 10^?12). Relationships between cell viability and weight of the donor fat pad were also discovered. Donor genetic background is a major determinant of in vitro ADSC phenotypes. This study supports the development of forward genetics strategies to identify genes that underlie ADSC phenotypic diversity, which will inform efforts to improve cell-based therapies.     

Correlation between preoperative magnetic resonance spectroscopic data on high grade gliomas and morphology of Ki-67-positive tumor cell nuclei

OBJECTIVE: To investigate possible statistical correlations between metabolic data from preoperative proton magnetic resonance spectroscopy (1HMRS) and morphology of proliferating tumor cell nuclei in anaplastic gliomas and glioblastomas. STUDY DESIGN: Ki-67-positive tumor cell nuclei in paraffin sections of surgical specimens from 36 patients (7 anaplastic gliomas, World Health Organization grade 3; 29 glioblastomas, World Health Organization grade 4) were investigated by means of a digital image analysis system. Stringent inclusion criteria were formulated for all cases with respect to histologic quality and spectroscopic examination. As morphometric variables, nuclear area, shape variables (roundness factor, size-invariate Fourier amplitudes) and density of Ki-67-positive tumor cell nuclei per reference area were determined. RESULTS: Correlation analysis according to Spearman revealed a significant positive correlation between the total creatine (TCR) peak and nuclear area (P = .005). This correlation was also found within the glioblastoma group (P = .019). There was also a significant negative correlation of nuclear area with the ratio between choline and TCR in all cases (P = .014) and within the glioblastoma group (P = .046). No significant correlation of spectroscopic data was found with nuclear shape or density of Ki-67-positive tumor cell nuclei. CONCLUSION: The results demonstrate a correlation between spectroscopic data and morphology of proliferating tumor cell nuclei (nuclear size) in high grade gliomas. This study is part of a detailed investigation of the interrelationship between preoperative 1HMRS and quantitative histomorphology of gliomas.     

Stimulatory effect of 17β-estradiol on osteogenic differentiation potential of rat adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ADSCs) are considered as a potential cell source for regenerative medicine and tissue engineering. Although ADSCs have greater proliferation capacity than bone marrow stem cells (BMSCs), lower differentiation ability of these cells limits their utility in experimental and clinical studies. The purpose of this study was to investigate whether 17β-estradiol (E(2)) has a stimulatory effect on osteogenic differentiation potential of ADSCs in vitro. ADSCs were isolated from visceral adipose tissues of rats and treated with different concentrations of E(2) in osteogenic medium (OM) for 21 days. The differences in osteogenic differentiation potential of the cultures were assessed by von Kossa staining, measurement of alkaline phosphatase (ALP) activity and calcium levels. ADSCs cultured in OM supplemented with E(2) showed greater bone-like nodule formation and mineral deposition in comparing with the cells grown in OM. In addition, ALP activity and calcium levels also were significantly higher in the cultures exposed to E(2) than the cells treated only with OM (p < 0.005, n = 5). Our results suggest that E(2) may stimulate the osteogenic differentiation of ADSCs and therefore, can be used as an inducing agent to improve the efficiency of these cells in in vitro and in vivo studies.