Reliable Source Of Stem Cells Research Articles

Comparison of the efficacy of bone morphogenetic protein-4 on in vitro differentiation of murine adipose and bone marrow mesenchymal stem cells into primordial germ cells.

Background and purpose:In vitro development of functional gametes from pluripotent stem cells is a promising prospect to treat infertility. Mesenchymal stem cells with a high degree of plasticity and less tumorigenicity are a reliable source of stem cells for the generation of gametes. The present study aimed to compare the differentiation potential in the mesenchymal stem cells that are derived from bone marrow (BMDMSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) into germ cells in a culture medium containing bone morphogenic protein-4 (BMP-4).Experimental approach:In this study, MSCs were isolated from both bone marrow and adipose tissue of murine samples. To further verify the nature of the harvested stem cells, their multipotency and surface marker were examined. The identified stem cells were cultured in a medium supplemented with 0 and 25 ng/mL of BMP-4 for 4 days. Flow cytometry analysis, immunofluorescence staining, and real RT-PCR were used to assess the expression levels in germ cell-specific biomarkers (Mvh, Dazl, Stra8, and Scp3).Findings/Results:CD44+, CD45-, CD31-, BMD-MSCs, and AD-MSCs showed to be capable of differentiating to osteo-adipogenic lineages. The flow cytometry, immunofluorescence, and RT-PCR results indicated that early germ cell markers (Mvh and Dazl) were expressed in both types of cells but they were significantly higher in BMD-MSCs than AD-MSCs.Conclusion and implications:Based on our results, the addition of exogenous BMP4 to the culture medium could differentiate BMD-MSCs and AD-MSCs into primordial germ cells, but it is inadequate to further develop into late germ cells in vitro. Moreover, the results revealed that, although AD-MSCs were easier to collect and had faster growth and proliferation rates than BMD-MSCs, the BMD-MSCs were better capable of differentiation into primordial germ cells. They may serve to be considered a more suitable source of MSC for in vitro generation of gametes than AD-MSCs.

Cell quality evaluation with gene expression analysis of spheroids (3D) and adherent (2D) adipose stem cells.

Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (SASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by "cell-quality"; for this reason, the involvement of stress signals and cellular aging need to be further investigated. Here, we performed a comparative analysis of genes connected with stemness, aging, telomeric length and oxidative stress, in 3D and 2D primary cultures. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P<0.001) in SASCs-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P<0.001). The 3D and 2D cultures also had a different gene expression profile for genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P<0.01 and P<0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P<0.05, P<0.01 and P<0.001) and presented a striking large variation in their cellular redox state. Based on our findings, we propose a "cell quality" model of SASCs, highlighting a precise molecular expression of several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.

Isolating and Characterizing Adipose-Derived Stem Cells.

The research and application of regeneration medicine will require a reliable source of stem cells. Adipose tissue has proven to be an easily accessible and rich source of adult stem cells, termed adipose-derived stem cells (ASCs). ASCs have the most important advantage over stem cells from other available sources. There is no other human tissue as abundant as adipose tissue, making it possible to isolate adequate numbers of ASCs for potential clinical applications. Here, we describe detailed methods for isolating and characterizing ASCs. These procedures can be applied to adipose tissue not only for humans but also for other species.

Platelet-Derived Growth Factor Receptor-Positive Pericytic Cells of White Adipose Tissue from Critical Limb Ischemia Patients Display Mesenchymal Stem Cell-Like Properties.

BackgroundThe pericytes in the blood vessel wall have recently been identified to be important in regulating vascular formation, stabilization, remodeling, and function. We isolated and identified pericyte-like platelet-derived growth factor receptor beta-positive (PDGFRβ+) cells from the stromal vascular fraction (SVF) of adipose tissue from critical limb ischemia (CLI) patients and investigated their potential as a reliable source of stem cells for cell-based therapy.MethodsDe-identified subcutaneous fat tissues were harvested after amputation in CLI patients. Freshly isolated SVF cells and culture-expanded adipose-derived stem cells (ADSCs) were quantified using flow cytometry. A matrigel tube formation assay and multi-lineage differentiation were performed to assess pericytic and mesenchymal stem cell (MSC)-like characteristics of PDGFRβ+ ADSCs.ResultsPDGFRβ+ cells were located in the pericytic area of various sizes of blood vessels and coexpressed mesenchymal stem cell markers. PDGFRβ+ cells in freshly isolated SVF cells expressed a higher level of stem cell markers (CD34 and CXCR4) and mesenchymal markers (CD13, CD44, CD54, and CD90) than PDGFRβ– cells. In vitro expansion of PDGFRβ+ cells resulted in enrichment of the perivascular mesenchymal stem-like (PDGFRβ+/CD90+/CD45–/CD31–) cell fractions. The Matrigel tube formation assay revealed that PDGFRβ+ cells were located in the peritubular area.ConclusionsPDGFRβ+ ADSCs cells demonstrated a good multilineage differentiation potential. Pericyte-like PDGFRβ+ cells from the SVF of adipose tissue from CLI patients had MSC-like characteristics and could be amplified by in vitro culture with preservation of their cell characteristics. We believe PDGFRβ+ cells in the SVF of adipose tissue can be used as a reliable source of stem cells even in CLI patients.

Human hair follicle stem cell differentiation into contractile smooth muscle cells is induced by transforming growth factor-β1 and platelet-derived growth factor BB

Smooth muscle cells (SMCs) are important in vascular homeostasis and disease and thus, are critical elements in vascular tissue engineering. Although adult SMCs have been used as seed cells, such mature differentiated cells suffer from limited proliferation potential and cultural senescence, particularly when originating from older donors. By comparison, human hair follicle stem cells (hHFSCs) are a reliable source of stem cells with multi-differentiation potential. The aim of the present study, was to develop an efficient strategy to derive functional SMCs from hHFSCs. hHFSCs were obtained from scalp tissues of healthy adult patients undergoing cosmetic plastic surgery. The hHFSCs were expanded to passage 2 and induced by the administration of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor BB (PDGF-BB) in combination with culture medium. Expression levels of SMC-related markers, including α-smooth muscle actin (α-SMA), α-calponin and smooth muscle myosin heavy chain (SM-MHC), were detected by immunofluorescence staining, flow cytometry analysis and reverse transcription-polymerase chain reaction (RT-PCR). When exposed to differentiation medium, hHFSCs expressed early, mid and late markers (α-SMA, α-calponin and SM-MHC, respectively) that were similar to the markers expressed by human umbilical artery SMCs. Notably, when entrapped inside a collagen matrix lattice, these SM differentiated cells showed a contractile function. Therefore, the present study developed an efficient strategy for differentiating hHFSCs into contractile SMCs by stimulation with TGF-β1 and PDGF-BB. The high yield of derivation suggests that this strategy facilitates the acquisition of the large numbers of cells that are required for blood vessel engineering and the study of vascular disease pathophysiology.

Isolation and Characterization of a Spontaneously Immortalized Multipotent Mesenchymal Cell Line Derived from Mouse Subcutaneous Adipose Tissue

The emerging field of tissue engineering and regenerative medicine is a multidisciplinary science that is based on the combination of a reliable source of stem cells, biomaterial scaffolds, and cytokine growth factors. Adult mesenchymal stem cells are considered important cells for applications in this field, and adipose tissue has revealed to be an excellent source of them. Indeed, adipose-derived stem cells (ASCs) can be easily isolated from the stromal vascular fraction (SVF) of adipose tissue. During the isolation and propagation of murine ASCs, we observed the appearance of a spontaneously immortalized cell clone, named m17.ASC. This clone has been propagated for more than 180 passages and stably expresses a variety of stemness markers, such as Sca-1, c-kit/CD117, CD44, CD106, islet-1, nestin, and nucleostemin. Furthermore, these cells can be induced to differentiate toward osteogenic, chondrogenic, adipogenic, and cardiogenic phenotypes. m17.ASC clone displays a normal karyotype and stable telomeres; it neither proliferates when plated in soft agar nor gives rise to tumors when injected subcutaneously in NOD/SCID-γ (null) mice. The analysis of gene expression highlighted transcriptional traits of SVF cells. m17.ASCs were genetically modified by lentiviral vectors carrying green fluorescent protein (GFP) as a marker transgene and efficiently engrafted in the liver, when injected in the spleen of NOD/SCID-γ (null) monocrotaline-treated mice. These results suggest that this non-tumorigenic spontaneously immortalized ASC line may represent a useful tool (cell model) for studying the differentiation mechanisms involved in tissue repair as well as a model for pharmacological/toxicological studies.

Haploidentical hematopoietic stem cell transplantation without in vitro T-cell-depletion for the treatment of hematologic diseases

Haploidentical hematopoietic stem cell transplantation (HSCT) has been accepted worldwide as an alternative treatment for patients with hematologic diseases who do not have a human leukocyte antigen (HLA) identical sibling donor or who require urgent transplantation. The results from our nine-year experience showed that granulocyte colony-stimulating factor (G-CSF) primed bone marrow (G-BM) combined with peripheral blood grafts (G-PB) from haploidentical donors, without in vitro T cell depletion (TCD), is a reliable source of stem cells for transplantation to cure acute leukemia and chronic myeloid leukemia. Recent findings confirmed that unmanipulated haploidentical HSCT is a promising protocol that can be successfully extended to treat intermediate and high-risk myelodysplastic syndrome and severe aplastic anemia. Recent observations suggest the association of improved immune recovery with better transplant outcomes after haploidentical HSCT. Chronic graft-vs.-host-disease severity strongly correlates with negative impacts on patients' health-related quality of life, suggesting that it should be successfully controlled.

Human Amnion–Derived Cells as a Reliable Source of Stem Cells

Human amnion-derived cells possess great potential for the repair of human neural disorders, and recent studies have broadened the spectrum for applications because they exhibit the characteristics of multipotent stem cells. These cells express embryonic stem cell markers such as Oct4, Nanog, Sox2, SSEA-3, SSEA-4 and Rex1, and can differentiate into multiple primary germ layers both in vitro and in vivo. Moreover, induced pluripotent stem cells have been generated from amnion-derived cells by virus-mediated delivery of three or four pluripotency-relating transcription factors or by the introduction of only one transcription factor with electroporation. Because human amnion-derived cells are readily available, less likely to contain genetic aberrations and can be reprogrammed earlier and more efficiently than differentiated cells, they can be ideal resources as the donor pluripotent stem cells for therapeutic purposes. We discuss here the highlights of recent studies and potential applications of human amnion-derived multipotent stem cells to stem cell biology as well as to regenerative medicine in the field of aging, heart disease, diabetes and neural disorders.

Long‐term follow‐up of haploidentical hematopoietic stem cell transplantation without in vitro T cell depletion for the treatment of leukemia

Many patients who require allogeneic hematopoietic stem cell transplantation (allo-HSCT) lack a human leukocyte antigen (HLA)-matched donor. Recently, a new strategy was developed for HLA-mismatched/haploidentical transplantation from family donors without in vitro T cell depletion (TCD). Over the past 9 years, 756 patients underwent haploidentical transplantation using a protocol developed by the authors, which combines granulocyte-colony stimulating factor-primed bone marrow (G-BM) and peripheral blood stem cells without in vitro TCD. The long-term outcome with this treatment modality was reported, and a risk-factor analysis was provided. Of these patients, 752 (99%) achieved sustained, full donor chimerism. The incidence of grades 2 through 4 acute graft-versus-host disease (GVHD) was 43%, and the 2-year cumulative incidence of total chronic GVHD was 53%. The 3-year cumulative incidence of nonrelapse mortality was 18%. The 2-year cumulative incidences of relapse were 15% and 26% in the standard-risk and high-risk groups, respectively. Of the 756 patients, 480 survived throughout the follow-up period of 1154 days (range: 335-3511 days) with the 3-year leukemia-free survival rates of 68% and 49% in the standard-risk and high-risk groups, respectively. Lower leukemia-free survival was associated with high-risk disease status (P = .001), chronic myelogenous leukemia disease type (P = .004), neutrophil engraftment beyond 13 days after transplant (P = .012), and the occurrence of grades 2 through 4 acute GVHD (P = .019). The results from the authors' 9-year experience showed that G-BM combined with peripheral blood stem cells from haploidentical donors, without in vitro TCD, is a reliable source of stem cells for transplantation by using the protocol developed by the authors.

Adipose-derived stem cells: Isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.

Adipose-Derived Stem Cells for Regenerative Medicine

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue represents an abundant and accessible source of adult stem cells with the ability to differentiate along multiple lineage pathways. The isolation, characterization, and preclinical and clinical application of adipose-derived stem cells (ASCs) are reviewed in this article.