Multilineage differentiation of muscle-derived stem cells from GFP transgenic mice

It is unclear whether green fluorescent protein (GFP) expression is maintained during the course of multilineage differentiation of muscle-derived stem cells (MDSCs). We isolated MDSCs from GFP-transgenic mice and transferred them to chondrogenic, neurogenic or myogenic media. Multilineage differentiation was examined by morphological observation, histological staining, immunocytochemical staining, real-time RT-PCR and Western blot. Both differentiated cells and non-differentiated cells maintained stable GFP expression until the cells exhibited a senescent phenotype. Thus, MDSCs from GFP-transgenic mice have multilineage potential in vitro and that GFP expression does not influence the multilineage potential of MDSCs (or vice versa).     

Rapid isolation of muscle-derived stem cells by discontinuous Percoll density gradient centrifugation

We investigated relationship between the maturity and density of muscle cells and developed a rapid isolation method to acquire stem cells from skeletal muscle. Mononuclear cells were isolated from the lower hind-limb muscles of 7-d-old male Sprague–Dawley rats and separated by Percoll density gradient centrifugation. After centrifugation, the cells were layered in the interfaces between each Percoll density layer. Flow cytometry was used to investigate the Sca-1, Pax7, CD34, CD45, M-cadherin, and myosin expression of the cells in each density layer. We found that CD45-positive cells were not present in freshly isolated muscle cells. CD34-, Pax7-positive cells were mainly observed at the interface between the 15% and 25% Percoll layers and had a density of 1.0235–1.0355 g/ml. Cells positive for M-cadherin were at the 25–35% Percoll density interface and had a density of 1.0355–1.0492 g/ml. We conclude that because there appears to be a correlation between maturity and density, muscle-derived stem cells may be isolated successfully from the 15–25% Percoll interface.     

Correction to: Multilineage differentiation of muscle-derived stem cells from GFP transgenic mice

Biotechnology Letters -     

An efficient method for isolation of murine bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been isolated based on the ability of adherence to plastic surfaces. The potential of these cells to differentiate along multiple lineages is the key to identifying stem cell populations in the absence of molecular markers. Here we describe a homogenous population of MSCs from mouse bone marrow isolated using a relatively straightforward and novel approach. This method is based on the combination of frequent medium change (FMC) and treatment of the primary cultures with trypsin. Cells isolated using this method demonstrated the MSCs characteristics including their ability to differentiate into mesenchymal lineages. MSCs retained the differentiation potentials in expanded cultures up to 10 passages. Isolated MSCs were reactive to the CD44, Sca-1, and CD90 cell surface markers. MSCs were negative for the hematopoietic surface markers such as CD34, CD11b, CD45, CD31, CD106, CD117 and CD135. The data presented in this report indicated that this method can result in efficient isolation of homogenous populations of MSCs from mouse bone marrow.     

Long-term self-renewal of postnatal muscle-derived stem cells

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.     

High efficient isolation and systematic identification of human adipose-derived mesenchymal stem cells

Background  

Developing efficient methods to isolate and identify human adipose-derived mesenchymal stem cells (hADSCs) remains to be one of the major challenges in tissue engineering.     

Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.     

Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin(+) cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34(+) cells and Bcl-2(+) cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34(+)Bcl-2(+) enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.     

A simple and efficient cryopreservation method for primate embryonic stem cells

Human embryonic stem (ES) cells have the potential to differentiate into all cell types. As these cells may be able to provide an unlimited cell source for transplantation therapies, it is necessary to establish reliable methods for their handling and manipulation, including human ES cell cryopreservation. Here, we report the development of a simple and efficient cryopreservation method for primate ES cell lines using vitrification in conventional cryovials. Using standard slow-rate cooling methods, the cryopreservation efficiency for cynomolgus monkey ES cell lines was approximately 0.4%, while that for a human ES cell line was virtually 0%. Primate ES cell lines, however, were successfully cryopreserved by the present vitrification method using conventional cryovials yielding a survival rate of about 6.5% for monkey ES cells and 12.2% for human ES cells. Vitrified ES cells quickly recovered after thawing and exhibited a morphology indistinguishable from non-vitrified cells. In addition, they retained a normal karyotype and continued to express ES cell markers after thawing. Thus, our vitrification ES cell cryopreservation method expands the utility of primate ES cells for various research and clinical purposes.     

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterization showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.     

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterisation showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.     

Selection of appropriate isolation method based on morphology of blastocyst for efficient derivation of buffalo embryonic stem cells

The efficiency of embryonic stem cell (ESC) derivation from all species except for rodents and primates is very low. There are however, multiple interests in obtaining pluripotent cells from these animals with main expectations in the fields of transgenesis, cloning, regenerative medicine and tissue engineering. Researches are being carried out in laboratories throughout the world to increase the efficiency of ESC isolation for their downstream applications. Thus, the present study was undertaken to study the effect of different isolation methods based on the morphology of blastocyst for efficient derivation of buffalo ESCs. Embryos were produced in vitro through the procedures of maturation, fertilization and culture. Hatched blastocysts or isolated inner cell masses (ICMs) were seeded on mitomycin-C inactivated buffalo fetal fibroblast monolayer for the development of ESC colonies. The ESCs were analyzed for alkaline phosphatase activity, expression of pluripotency markers and karyotypic stability. Primary ESC colonies were obtained after 2–5 days of seeding hatched blastocysts or isolated ICMs on mitomycin-C inactivated feeder layer. Mechanically isolated ICMs attached and formed primary cell colonies more efficiently than ICMs isolated enzymatically. For derivation of ESCs from poorly defined ICMs intact hatched blastocyst culture was the most successful method. Results of this study implied that although ESCs can be obtained using all three methods used in this study, efficiency varies depending upon the morphology of blastocyst and isolation method used. So, appropriate isolation method must be selected depending on the quality of blastocyst for efficient derivation of ESCs.     

A rapid and efficient method for primary culture of human adipose-derived stem cells

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.     

Embryo sac isolation inArabidopsis thaliana: A simple and efficient technique for structure analysis and mutant selection

Arabidopsis thaliana has been widely used as a model plant in gene function analysis. However, its tiny flower and curved embryo sac make it difficult to study gene expression during megagametogenesis, fertilization, and early embryogenesis, especially in the screening of mutants from those developmental processes. The techniques currently available are sectioning and whole-mount clearing of ovules; however, sectioning is time consuming and laborious for quantitative analysis, and whole-mount clearing, makes clear cytological observation impossible. Reported here is a simple and efficient method based on enzymatic isolation of embryo sacs that enables both quantitative analysis and elaborate cytological observation for gene expression investigation and mutant screening.     

Bone marrow mesenchymal stem cells modified by angiogenin-1 promotes tissue repair in mice with oxygen-induced retinopathy of prematurity by promoting retinal stem cell proliferation and differentiation

Retinopathy has become one of the major factors that lead to blindness worldwide. Although many clinical therapies are concerned about such disease, most of them focus on symptoms alleviation. In this study, we aim to investigate whether coculture retinal stem cells (RSCs) with bone marrow mesenchymal stem cells transfected with angiogenin-1 (Ang-1-BMSCs) affects the damaged retinal tissue of oxygen-induced retinopathy of prematurity (OIR-ROP) mice. After OIR-ROP mouse model establishment, Ang-1-BMSCs, RSCs, and OIR-ROP retinal tissues were cocultured in a a transwell chamber. RSCs proliferation and the expression of Ang-1, insulin-like growth factor-1 (IGF-1) in the supernatant of RSCs, as well as β-tubulin and protein kinase C (PKC) expression were evaluated. Finally, the repair of OIR-ROP mice retinal tissues was observed by injecting Ang-1-BMSCs + RSCs. In the OIR-ROP mouse model, RSCs cocultured with OIR-ROP retinal tissues could be induced to differentiate into cells expressing β-tubulin and PKC and promote the expression of Ang-1 and IGF-1. coculture of Ang-1-BMSCs further enhanced the proliferation and differentiation of RSCs by promoting the expression of Ang-1 and IGF-1. Coculture of RSCs + Ang-1-BMSCs induced differentiation of Ang-1-BMSCs through interaction among intercellular factors and restored the damaged retinal tissue of OIR-ROP mice. Collectively, our study provided evidence that coculture of Ang-1-BMSCs and RSCs could promote the proliferation and differentiation of RSCs and improve the treatment for the damaged retina tissue of OIR-ROP mice.