Characterization and comparison of adipose tissue‐derived cells from human subcutaneous and omental adipose tissues

Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue‐derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31^?CD34⁺CD45^?CD90^‐CD105^?CD146⁺ population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31^?CD34⁺CD45^?CD90^?CD105^?CD146^? population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood‐derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose‐derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Human CD34+/CD90+ ASCs Are Capable of Growing as Sphere Clusters,Producing High Levels of VEGF and Forming Capillaries

Background

Human adult adipose tissue is an abundant source of mesenchymal stem cells (MSCs). Moreover, it is an easily accessible site producing a considerable amount of stem cells.

Methodology/Principal Findings

In this study, we have selected and characterized stem cells within the stromal vascular fraction (SVF) of human adult adipose tissue with the aim of understanding their differentiation capabilities and performance. We have found, within the SVF, different cell populations expressing MSC markers – including CD34, CD90, CD29, CD44, CD105, and CD117 – and endothelial-progenitor-cell markers – including CD34, CD90, CD44, and CD54. Interestingly, CD34⁺/CD90⁺ cells formed sphere clusters, when placed in non-adherent growth conditions. Moreover, they showed a high proliferative capability, a telomerase activity that was significantly higher than that found in differentiated cells, and contained a fraction of cells displaying the phenotype of a side population. When cultured in adipogenic medium, CD34⁺/CD90⁺ quickly differentiated into adipocytes. In addition, they differentiated into endothelial cells (CD31⁺/VEGF⁺/Flk-1⁺) and, when placed in methylcellulose, were capable of forming capillary-like structures producing a high level of VEGF, as substantiated with ELISA tests.

Conclusions/Significance

Our results demonstrate, for the first time, that CD34⁺/CD90⁺ cells of human adipose tissue are capable of forming sphere clusters, when grown in free-floating conditions, and differentiate in endothelial cells that form capillary-like structures in methylcellulose. These cells might be suitable for tissue reconstruction in regenerative medicine, especially when patients need treatments for vascular disease.     

Identification of active and quiescent adipose vascular stromal cells

Background aimsRecent studies have demonstrated the existence of both active and quiescent stem cells in bone marrow, hair follicle and intestine. We attempted to identify active and quiescent vascular stromal cells (VSC) in adipose tissue.MethodsFor identification of active VSC, adult rats were injected intraperitoneally with thymidine analog 5-ethynyl-2-deoxyuridine (EdU) and their subcutaneous tissue harvested 3 days later. For identification of quiescent VSC, newborn rats were injected intraperitoneally with EdU and their subcutaneous tissue harvested 9 weeks later. The harvested adipose tissues were examined for the co-localization of EdU with VSC marker CD34, smooth muscle marker SMA, endothelial marker RECA and pericyte marker CD140b.ResultsIn adult rat adipose tissues harvested 3 days after EdU injection, there were 28.80 ± 8.70 (mean ± SD) EdU⁺ cells/100 × microscopic field, and approximately 6.2% of cell nuclei were labeled with EdU. The percentages of EdU⁺ cells expressing the following markers were approximately: 84 for CD34, 5.6 for RECA (rat endothelial marker), 3.7 for SMA and 14.8 for CD140b. In the adipose tissues of newborn rats that were harvested 9 weeks after EdU injection, the percentages of EdU⁺ cells expressing the following markers were approximately: 76 for CD34, 1.8 for RECA, 0 for SMA and 12.9 for CD140b. In both the short-term (active) and long-term (quiescent) EdU-labeled adipose tissues, the EdU label was consistently co-localized with CD34 and in the proximity of CD140b stain or in the adventitia.ConclusionsBoth active and quiescent VSC expressed CD34 and localized to capillaries and the adventitia of larger blood vessels.     

Hematopoietic stem cell and mesenchymal stem cell population size in bone marrow samples depends on patient’s age and harvesting technique

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45^?/CD271⁺ cells correlates better with CFU-F numbers than the population of the CD45^?/CD73⁺/CD90⁺/CD105⁺ cells. The relationship between the hematopoietic CD45^dim/CD34⁺ cell concentration and mesenchymal CFU-Fs or CD45^?/CD271⁺ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45^dim/CD34⁺, mesenchymal CD45^?/CD73⁺/CD90⁺/CD105⁺ and CD45^?/CD271⁺ stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

CD106 Identifies a Subpopulation of Mesenchymal Stem Cells with Unique Immunomodulatory Properties

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106⁺cells and CD106⁻cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106⁺CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106⁺CV-MSCs expressed more cytokines than CD106⁻CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.     

CD4+CD25+ regulatory T cells are not required for mesenchymal stem cell function in fully MHC-mismatched mouse cardiac transplantation

Although the immunomodulative properties of mesenchymal stem cells (MSCs) open up attractive possibilities in solid-organ transplantation, information concerning the optimal dose, route, timing of administration, major histocompatibility complex (MHC)-restriction and relevant mechanisms is currently lacking. Therefore, better characterization of MSC immunoregulatory activity and elucidation of its mechanisms are crucial. In this study, we confirmed that MSCs did not elicit proliferation by allogeneic CD4⁺ T cells, suggesting that MSCs were not immunogenic. By using C57BL/6 mouse MSCs as donor-derived or recipient-derived or as third-party MSCs, we discovered that MSCs suppressed CD4⁺ T cell proliferation and prolonged mouse cardiac allograft survival in a dose-dependent and non-MHC-restricted manner. We also found that intraperitoneal administration favored survival prolongation, although this prolongation was weaker than that via the intravenous route. Only infusion at earlier time points favored survival prolongation. Depletion of CD4⁺CD25⁺ T cells did not affect the immunosuppression of MSCs on CD4⁺ T cells. Moreover, MSCs did not induce regulatory T cells. The in vivo data revealed that MSCs did not increase the percentage of CD4⁺CD25⁺ T cells and FoxP3 expression. More importantly, we demonstrated for the first time that depletion of CD4⁺CD25⁺ T cells did not hinder MSC-induced survival prolongation, indicating that CD4⁺CD25⁺ regulatory T cells were not essential for the prolongation of MSC-mediated allograft survival.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro

To investigate the differentiation potential of purified CD90⁺ cells sorted from adipose-derived stem cells (ADSCs), CD90⁺ cells were sorted from rabbit ADSCs using flow cytometry. Then, cell expansion of CD90⁺ cells and unsorted ADSCs was observed using an inverted microscope. Furthermore, cell surface markers including CD40, CD105, and CD90 on CD90⁺ cells and unsorted ADSCs were quantified using flow cytometry. Additionally, multi-lineage differentiation ability between CD90⁺ cells and unsorted ADSCs was compared, and expression of adipocyte-related genes PPAR-r and CEBPA as well as stem cell-related gene SOX2 in CD90⁺ cells and unsorted ADSCs was determined using real-time quantitative PCR. We found that CD90⁺ cells had a stronger cell proliferation ability than unsorted ADSCs. CD90⁺ cells showed a stronger ability of osteoblast and chondrocyte differentiation than unsorted ADSCs and CD90^? cells, whereas the adipose differentiation ability of CD90⁺ cells was similar to that of ADSCs and CD90^? cells. CD14, CD105, and CD90 on CD90⁺ cells were expressed more highly than those on ADSCs. Additionally, the mRNA expression level of SOX2 in CD90⁺ cells was significantly higher than that in ADSCs, whereas the expression of PPAR-r and CEBPA was markedly lower than that in ADSCs. These results suggested that the purified CD90⁺ cells sorted from ADSCs exhibit a stronger differentiation potential than the unsorted ADSCs.     

Mesenchymal stem cells and their use in therapy: What has been achieved?

The considerable therapeutic potential of human multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) has generated increasing interest in a wide variety of biomedical disciplines. Nevertheless, researchers report studies on MSCs using different methods of isolation and expansion, as well as different approaches to characterize them; therefore, it is increasingly difficult to compare and contrast study outcomes. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed minimal criteria to define human MSCs. First, MSCs must be plastic-adherent when maintained in standard culture conditions (α minimal essential medium plus 20% fetal bovine serum). Second, MSCs must express CD105, CD73 and CD90, and MSCs must lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules. Third, MSCs must differentiate into osteoblasts, adipocytes and chondroblasts in vitro. MSCs are isolated from many adult tissues, in particular from bone marrow and adipose tissue. Along with their capacity to differentiate and transdifferentiate into cells of different lineages, these cells have also generated great interest for their ability to display immunomodulatory capacities. Indeed, a major breakthrough was the finding that MSCs are able to induce peripheral tolerance, suggesting that they may be used as therapeutic tools in immune-mediated disorders. Although no significant adverse events have been reported in clinical trials to date, all interventional therapies have some inherent risks. Potential risks for undesirable events, such as tumor development, that might occur while using these stem cells for therapy must be taken into account and contrasted against the potential benefits to patients.     

Isolation of adipose tissue mesenchymal stem cells without tissue destruction: A non-enzymatic method

The conventional enzymatic method is widely used for mesenchymal stem cells (MSCs) isolation from adipose tissue. The method holds major drawbacks; it is costly, time-consuming and results in a heterogeneous cell population. Besides, digestion of extracellular matrix causes cell injury and compromise proliferation and differentiation of the cells. Also, because of over handling the samples are also prone to contamination. Here, we introduce a non-enzymatic method for MSCs isolation without disturbing the cells habitat. Small pieces of adipose tissue obtained from animal or human liposuction were explanted into a culture flask, immobilized by fetal bovine serum (FBS) and incubated overnight. The explants were then irrigated with DMEM containing FBS. Within few days, the fibroblast-like cells migrated from the tissue and proliferated rapidly. When subconfluent, the cells were harvested, expanded through 3 passages and used for immunophenotyping and differentiation assays. As judged by flow cytometric analysis of surface markers (CD44⁺, CD105⁺, CD34⁻, CD45⁻), Oil Red O and Alizarin Red staining, the MSCs isolated by our non-enzymatic method were pluripotent and exhibited the potential for differentiation into adipocyte and osteoblast. Great isolation yields, homogeneity of isolated cells, brief procedure, and high economy are the advantages of our method over the conventional protocol.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Derivation of multipotent nestin+/CD271−/STRO-1− mesenchymal-like precursors from human embryonic stem cells in chemically defined conditions

The successful establishment of stem cell-based therapies requires multipotent, immunocompatible stem cells, highly efficient strategies for direct differentiation, and most importantly, optimal culture conditions for large-scale expansion of such cell populations. Other than adult tissues, human embryonic stem cells (hESCs) represent another infinitely expansible source for mesenchymal stem cell (MSC) derivation. Here, we reproducibly derived a population of Nestin⁺/CD271^?/STRO-1^? mesenchymal-like precursors from hESCs (hESC-MPs) in chemically defined conditions, without requiring any serum or serum replacement of animal origin, based on a Y-27632-assisted monolayer culture system. These cells showed slim fibroblastic morphology, and satisfied the criteria of MSCs including self-renewal, the expression of multiple MSC-specific markers and the ability to differentiate into osteoblasts, adipocytes and chondrocytes. Compared with previously reported hESC-derived MSCs, our hESC-MPs were more multipotent, and could differentiate into representative derivatives of all three embryonic germ layers including mature smooth muscle cells, cardiomyocytes, functional hepatocytes and neural cells expressing various neurotransmitter phenotypes, making them an attractive cell source for regenerative medicine.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.