Clonal growth and differentiation of mesenchymal stromal cells from rat liver at different stages of embryogenesis

Fetal liver, during its hematopoietic activity, contains mesenchymal stromal cells (MSCs) generating its hematopoietic microenvironment. These cells are clonogenic and capable of multilineage differentiation; however, little is known about how their properties alter during embryogenesis. We compared the cloning efficiency of MSCs from rat fetal liver at 14, 16, and 20 days of development, as well as their capacity for osteo- and adipogenesis in vitro and chondrogenesis in vivo by ectopic transplantation of intact liver. The relative content of clonogenic MSCs in liver cell suspension was highest in 16-day fetuses and lowest in 20-day fetuses. Cells from 14-day fetuses exhibited high osteogenic and less apparent adipogenic and chondrogenic potential; cells from 20-day fetuses displayed weak adipogenic capacity and no osteo- or chondrogenic ability. These results show the correlation of MSC content and the cell differentiation potential with hematopoietic dynamics in developing rat liver. It may be thought that the changes we observed are related to the loss of hematopoietic activity and liver getting of definitive functions.     

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.     

Human and animal mesenchymal progenitor cells from bone marrow: Identification of serum for optimal selection and proliferation

Summary An undifferentiated subset of cells within the stromal cell population of bone marrow in postnatal mammals retains the capacity to differentiate along osteogenic, adipogenic, fibroblastic, and chondrogenic lines. These cells, which are referred to as mesenchymal stem cells (MSCs), can be maintainedin vitro and expanded in number through a process of subculturing. MSCs are maintained in culture in medium supplemented with 10% fetal bovine serum (FBS). It is believed that certain, as yet unidentified, serum components play critical roles in the attachment and proliferation of MSCs. Commercially available FBS is poorly characterized and may vary in composition and quality from lot to lot. This study describes a method for the selection of lots of FBS that best support maintenance of the undifferentiated state, mitotic expansion of MSCsin vitro, and retention of multilineage developmental potential in response to appropriate cues.     

Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.     

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.     

Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers

We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2-12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints.     

Long bone mesenchymal stem cells (Lb-MSCs): clinically reliable cells for osteo-diseases

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer–irrigator–aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7–10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.     

Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.     

Mesenchymal stem cells in the umbilical cord: phenotypic characterization, secretome and applications in central nervous system regenerative medicine

Mesenchymal Stem Cells (MSCs), have been defined and characterized by: 1) their ability to adhere to plastic culture flasks; 2) the positive expression of CD105, CD73, CD90 membrane antigens, and the lack of expression of others (e.g CD45 and CD34) and 3) the ability of differentiation under adequate conditions along the osteogenic, chondrogenic and adipogenic lineages. In recent years cells with these characteristics have been isolated from the Wharton Jelly (WJ) of the Umbilical Cord (UC). Similarly to bone marrow MSCs they have shown multilineage differentiation potential and to be able to provide trophic support to neighboring cells. According to the literature, there are two main populations of cells with a mesenchymal character within the human UC: Wharton's Jelly Mesenchymal Stem Cells (WJ-MSCs) and Human Umbilical Cord Perivascular Cells (HUCPVCs). In the present work our aim is to make a comprehensive review on MSCs populations of the WJ and how these cell populations may be used for future applications in CNS regenerative medicine. Following a brief insight on the general characteristics of MSC like cells, we will discuss the possible sources of stem cells within the WJ and the cord itself (apart UC blood), as well as their phenotypic character. As it has already been shown that these cells hold a strong trophic support to neighbouring cell populations, we will then focus on their secretome, namely which molecules have already been identified within it and their role in phenomena such as immunomodulation. The possible applications of these cells populations to CNS regenerative medicine will be addressed by critically reviewing the work that has been performed so far in this field. Finally, a brief insight will be made on what in the authors' opinion are the major challenges in the field for the future application of these cell populations in CNS regenerative medicine.     

Comparative study of clonal growth and differentiation of mesenchymal stromal cells from rat fetal liver at different developmental stages

Fetal liver during period of its hematopoietic activity contains mesenchymal stromal cells (MSC) that are known to play a major role in establishing hematopoietic microenvironment. These cells are capable of clonal growing and multilineage differentiation, but only limited data exist about changes in their properties during prenatal development. We compared cloning efficiency of MSC from liver of 14, 16 and 20 day rat fetuses and evaluated their potentials to in vitro osteo- and adipogenesis and in vivo chondrogenesis after whole organ ectopic transplantation. Content of clonogenic MSC in suspension of liver cells was maximal in 16 day fetuses and to a lesser extent in 20 day ones. MSC derived from 16 day fetuses demonstrated maximal potential to estimated lineages. Osteogenic potential of MSC from 14 day fetuses was comparable to whereas their adipogenic and chondrogenic abilities were inferior to that from 16 day fetuses. Cells from 20 day fetuses had only weak adipogenic potency and failed to differentiate into osteogenic of chondrogenic pathways. The results indicate that both number and differentiation potential of MSC in developing rat liver correlate with dynamics of hematopoiesis in this organ. Detected changes may be ascribed to the decline of hematopoiesis in liver and acquisition its definitive functions.     

再生障碍性贫血患者骨髓间充质干细胞生物学特性的初步研究

目的:研究再生障碍性贫血(aplasticanemia,从)患者骨髓间充质干细胞(mesenchymalstemcells,MSCs)的生物学特性和初步探讨其异常和AA发生的可能关系。方法:取AA患者骨髓间充质干细胞,测定其生长曲线和倍增时间;流式细胞仪检测其细胞周期和免疫表型;体外定向诱导其向脂肪、成骨、内皮和神经细胞分化;用real-timePCR及油红O染色法比较AA和正常对照组MSCs的成脂分化的不同。结果:AA患者和正常成人的MSCs均呈梭形贴壁生长;AA组细胞倍增时间长于对照组;CD105、CD44、CD29、CD106、FlK-1均阳性;96．51％的细胞处在G0／G1期;AA患者的MSCs保持了多向分化潜能,体外诱导形成脂滴较对照组早,诱导早期的脂蛋白脂酶表达增高。结论:再生障碍性贫血患者的骨髓间充质干细胞增殖能力较正常成人弱,骨髓间充质干细胞的易成脂性可能参与了再障的发病环节。     

Expansion on specific substrates regulates the phenotype and differentiation capacity of human articular chondrocytes

In this study, we investigated if monolayer expansion of adult human articular chondrocytes (AHAC) on specific substrates regulates cell phenotype and post-expansion multilineage differentiation ability. AHAC isolated from cartilage biopsies of five donors were expanded on plastic dishes (PL), on dishes coated with collagen type II (COL), or on slides coated with a ceramic material (Osteologic, OS). The phenotype of expanded chondrocytes was assessed by flow cytometry and real-time RT-PCR. Cells were then cultured in previously established conditions promoting differentiation toward the chondrogenic or osteogenic lineage. AHAC differentiation was assessed histologically, biochemically, and by real-time RT-PCR. As compared to PL-expanded AHAC, those expanded on COL did not exhibit major phenotypic changes, whereas OS-expanded cells expressed (i) higher bone sialoprotein (BSP) (22.6-fold) and lower collagen type II (9.3-fold) mRNA levels, and (ii) lower CD26, CD90 and CD140 surface protein levels (1.4-11.1-fold). Following chondrogenic differentiation, COL-expanded AHAC expressed higher mRNA levels of collagen type II (2.3-fold) and formed tissues with higher glycosaminoglycan (GAG) contents (1.7-fold), whereas OS-expanded cells expressed 16.5-fold lower collagen type II and generated pellets with 2.0-fold lower GAG contents. Following osteogenic differentiation, OS-expanded cells expressed higher levels of BSP (3.9-fold) and collagen type I (2.8-fold) mRNA. In summary, AHAC expansion on COL or OS modulated the de-differentiated cell phenotype and improved the cell differentiation capacity respectively toward the chondrogenic or osteogenic lineage. Phenotypic changes induced by AHAC expansion on specific substrates may mimic pathophysiological events occurring at different stages of osteoarthritis and may be relevant for the engineering of osteochondral tissues.     

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-γ2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.