Retinal stem cells: promising candidates for retina transplantation

Stem cell transplantation is widely considered as a promising therapeutic approach for photoreceptor degeneration, one of the major causes of blindness. In this review, we focus on the biology of retinal stem cells (RSCs) and progenitor cells (RPCs) isolated from fetal, postnatal, and adult animals, with emphasis on those from rodents and humans. We discuss the origin of RSCs/RPCs, the markers expressed by these cells and the conditions for the isolation, culture, and differentiation of these cells in vitro or in vivo by induction with exogenous stimulation. Commercial disclosure: none.     

Skeletal muscle differentiation potential of human adult bone marrow cells

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.     

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.     

The frequency, growth kinetics, and osteogenic/adipogenic differentiation properties of canine bone marrow stromal cells

Bone marrow stromal cells (BMSCs) have gained considerable attention as a potential source for cell transplantation therapies for a variety of diseases due to their accessibility, proliferative capacity, and multilineage differentiation properties. Canine BMSCs have been shown to contribute to regeneration of osseous tissues, but knowledge about their biology is currently limited. In the present study, we investigated the frequency of adult canine BMSCs in bone marrow, morphological features, growth kinetics, and osteogenic as well as adipogenic differentiation properties in vitro. Our data suggest that adult canine bone marrow contains approximately one BMSC in every 2.38 × 10⁴ bone marrow mononucleated cells (0.0042 ± 0.0019%, n = 5). Primary BMSC cultures consisted of morphologically heterogeneous adherent cell populations from which spindle-shaped cells grew and became the predominant cell type. Growth kinetics patterns were dependent on the initial cell seeding densities, resulting in the highest fold increase at lower cell density. In the presence of osteogenic and adipogenic inducers, primary BMSCs underwent morphological and phenotypic changes characteristic of osteogenic and adipogenic differentiation, respectively. This study provides insights into basic characterization of adult canine BMSCs.     

Redifferentiation of dedifferentiated chondrocytes and chondrogenesis of human bone marrow stromal cells via chondrosphere formation with expression profiling by large-scale cDNA analysis

Characterization of dedifferentiated chondrocytes (DECs) and mesenchymal stem cells capable of differentiating into chondrocytes is of biological and clinical interest. We isolated DECs and bone marrow stromal cells (BMSCs), H4-1 and H3-4, and demonstrated that the cells started to produce extracellular matrices, such as type II collagen and aggrecan, at an early stage of chondrosphere formation. Furthermore, cDNA sequencing of cDNA libraries constricted by the oligocapping method was performed to analyze difference in mRNA expression profiling between DECs and marrow stromal cells. Upon redifferentiation of DECs, cartilage-related extracellular matrix genes, such as those encoding leucine-rich small proteoglycans, cartilage oligomeric matrix protein, and chitinase 3-like 1 (cartilage glycoprotein-39), were highly expressed. Growth factors such as FGF7 and CTGF were detected at a high frequency in the growth stage of monolayer stromal cultures. By combining the expression profile and flow cytometry, we demonstrated that isolated stromal cells, defined by CD34(-), c-kit(-), and CD140alpha(- or low), have chondrogenic potential. The newly established human mesenchymal cells with expression profiling provide a powerful model for a study of chondrogenic differentiation and further understanding of cartilage regeneration in the means of redifferentiated DECs and BMSCs.     

Bone marrow mesenchymal stem cells differentiate into urothelial cells and the implications for reconstructing urinary bladder mucosa

To determine the ability of cultured bone marrow-derived mesenchymal stem cells (BMSCs) to differentiate into functional urothelium. BMSCs were isolated from the long bones of aborted fetal limbs by Percoll density gradient centrifugation and characterized by flow cytometry. Human fetal urinary bladders were cut into small pieces and cultured for 3–5 days until the growth of urothelial cells was established. BMSCs were then cocultured with neonatal urothelial cells and subsequently evaluated for antigen expression and ultramicrostructure, by immunocytochemistry and electron microscopy, respectively. A subset of BMSCs expressed the differentiation marker CD71. The BMSC markers CD34, CD45, and HLA-DR were barely detectable, confirming that these cells were not derived from hematopoietic stem cells or differentiated cells. In contrast, the stem cell markers CD29, CD44, CD105, and CD90 were highly expressed. BMSCs possessed the ability to differentiate into a variety of cellular subtypes, including osteocytes, adipocytes, and chondrocytes. The shapes of BMSCs changed, and the size of the cells increased, following in vitro coculture with urothelial cells. After 2 weeks of coculture, immunostaining of the newly differentiated BMSCs positively displayed the urothelial-specific keratin marker. Electron microscopy revealed that the cocultured BMSCs had microstructural features characteristic of epithelial cells. Pluripotent BMSCs can transdifferentiate into urothelial cells in response to an environment conditioned by neonatal urothelial cells, providing a means for the time-, labor- and cost-effective reconstruction of urinary bladder mucosa.     

Mesenchymal stem cells showed the highest potential for the regeneration of injured liver tissue compared with other subpopulations of the bone marrow

We have previously reported that bone marrow cells (BMCs) participate in the regeneration after liver injury. However, it is not established that this is the result of differentiation of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) or the combination of both. We investigated the contribution of each cell fraction to the regenerative process. First, we confirmed that transplanted stem cells migrate directly to injured liver tissue without dispersing to other organs. Next, we divided green fluorescent protein (GFP)-expressing BMCs into three populations as mononuclear cells, MSCs and HSCs. We then compared the engraftment capacity after transplantation of each fraction of cells into liver-injured mice. Of these, the MSCs transplanted group showed the highest GFP fluorescence intensities in liver tissue by flow cytometry analysis and confocal microscopic observation. Furthermore, MSCs showed differentiation potential into hepatocytes when co-cultured with injured liver cells, which suggests that MSCs showed highest potential for the regeneration of injured liver tissue compared with those of the other two cell refractions.     

人骨髓间充质干细胞脑内移植对食蟹猴脑出血的治疗作用

目的评价人骨髓间充质干细胞脑内移植对食蟹猴脑出血模型的治疗作用。方法符合普通级标准的成年食蟹猴12只,用自体股动脉抗凝血脑内注射方法建模后1周,用脑立体定位法在血肿周围植入人骨髓间充质干细胞,细胞数分别为高剂量5×106、低剂量1×106、对照组等体积生理盐水。利用MRI、PET、神经功能缺损评分和组织病理学对干细胞移植效果进行评价。结果神经功能评分显示干细胞移植1周后动物神经功能明显改善。PET结果显示干细胞移植后2周高剂量组血肿周围皮层、基底节核团的SUV%值与对照组间存在显著性差异(P=0.02)。移植后3周高、低剂量组血肿周围皮层、基底节核团的SUV%值与对照组间差异存在显著性(P值分别为0.03和0.04)。MRI显示剂量组血肿吸收速度大于对照组。病理检查可见剂量组坏死灶面积小于对照组,出血灶周围有大量新生血管生成,剂量组与对照组间差异存在显著性(P<0.01)。结论在损伤脑组织周围移植hBMSC可促进食蟹猴损伤神经组织的恢复,为hBMSC治疗脑出血的临床应用提供了重要实验依据。     

An overview and synopsis of techniques for directing stem cell differentiation in vitro

The majority of studies on stem cell differentiation have so far been based in vivo, on live animal models. The usefulness of such models is limited, since it is much more technically challenging to conduct molecular studies and genetic manipulation on live animal models compared to in vitro cell culture. Hence, it is imperative that efficient protocols for directing stem cell differentiation into well-defined lineages in vitro are developed. The development of such protocols would also be useful for clinical therapy, since it is likely that the transplantation of differentiated stem cells would result in higher engraftment efficiency and enhanced clinical efficacy, compared to the transplantation of undifferentiated stem cells. The in vitro differentiation of stem cells, prior to transplantation in vivo, would also avoid spontaneous differentiation into undesired lineages at the transplantation site, as well as reduce the risk of teratoma formation, in the case of embryonic stem cells. Hence, this review critically examines the various strategies that could be employed to direct and control stem cell differentiation in vitro.     

Myocardial regeneration potential of adipose tissue-derived stem cells

Various tissue resident stem cells are receiving attention from basic scientists and clinicians as they hold promise for myocardial regeneration. For practical reasons, adipose tissue-derived stem cells (ASCs) are attractive cells for clinical application in repairing damaged myocardium based on the following advantages: abundant adipose tissue in most patients and easy accessibility with minimally invasive lipoaspiration procedure. Several recent studies have demonstrated that both cultured and freshly isolated ASCs could improve cardiac function in animal model of myocardial infarction. The mechanisms underlying the beneficial effect of ASCs on myocardial regeneration are not fully understood. Growing evidence indicates that transplantation of ASCs improve cardiac function via the differentiation into cardiomyocytes and vascular cells, and through paracrine pathways. Paracrine factors secreted by injected ASCs enhance angiogenesis, reduce cell apoptosis rates, and promote neuron sprouts in damaged myocardium. In addition, Injection of ASCs increases electrical stability of the injured heart. Furthermore, there are no reported cases of arrhythmia or tumorigenesis in any studies regarding myocardial regeneration with ASCs. This review summarizes the characteristics of both cultured and freshly isolated stem cells obtained from adipose tissue, their myocardial regeneration potential, and the underlying mechanisms for beneficial effect on cardiac function, and safety issues.     

Stem cells with multilineage potential derived from porcine skin

Stem cells from farm animals are valuable cell models for the study of development, differentiation, and are potential efficient donors for nuclear transfer. Here we report the isolation and characterization of stem cells from porcine skin. These porcine skin-originated sphere (PSOS) cells expressed the neural progenitor marker, nestin, as well as genes that are critical for pluripotency such as Oct4 and Stat3. The PSOS cells proliferated actively in vitro and retained normal karyotype after long-term culture. When cultured in defined medium, they generated cells with characteristics of neurons and astrocytes. A subpopulation of cells differentiated into adipocyte-like cells when cultured in 10% fetal bovine serum. Clonal study demonstrated that PSOS exhibited clonal-generating capability. Clonal populations from individual stem cells could form neuron-, astrocyte-, and adipocyte-like cells upon inducted differentiation. Our findings represent the first report of skin-originated stem cells isolated from non-rodent animals.     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。     

Effects of autologous stem cell transplantation on ventricular electrophysiology in doxorubicin-induced heart failure

To investigate whether stem cell transplantation affects ventricular electrophysiology in vivo, either autologous bone marrow mesenchymal stem cells or skeletal myoblast cells were transplanted via a catheter into a doxorubicin-treated failing heart. Four weeks after transplantation, electrophysiological investigation showed that transplantation of either cell type prolonged the local activation time and increased the activation time dispersion. In the stem cell transplantation groups, a positive correlation was demonstrated between activation time dispersion and the number of stem cell-derived cells in the pacing site. It is concluded that transplantation of either mesenchymal stem cells or skeletal myoblast cells might exacerbate abnormalities of local ventricular conduction in the doxorubicin-treated failing heart.     

Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.     

Human astrocytes can be induced to differentiate into cells with neuronal phenotype

Several recent studies have proposed that astrocytes may contribute to neurogenesis, not only as a source of trophic substances regulating it, but also as stem cells themselves. In order to better understand these mechanisms, primary astrocyte cultures were established from human fetal brain. After 3-4 weeks in culture, astrocytes (about 95% GFAP+; neurofilament, NF-; neuro-specific enolase, NSE-) were treated with a cocktail of protein kinase activators and FGF-1. After 5 h of treatment, most cells showed morphological changes that increased progressively up to 24-48 h, exhibiting a round cell body with long processes. Immunocytochemistry showed that treatment-induced NF and NSE expression in about 40% of cells. Nestin expression increased after treatment, whereas GFAP immunostaining was not significantly modified. Western blot and RT-PCR confirmed the results. No neuronal electrophysiological properties were observed after treatment, suggesting an incomplete maturation under these experimental conditions. Understanding the regenerative capability and neurogenic potential of astrocytes might be useful in devising therapeutic approaches for a variety of neurological disorders.     

Type I Transglutaminase Accumulation in the Endoplasmic Reticulum May Be an Underlying Cause of Autosomal Recessive Congenital Ichthyosis

Type I transglutaminase (TG1) is an enzyme that is responsible for assembly of the keratinocyte cornified envelope. Although TG1 mutation is an underlying cause of autosomal recessive congenital ichthyosis, a debilitating skin disease, the pathogenic mechanism is not completely understood. In the present study we show that TG1 is an endoplasmic reticulum (ER) membrane-associated protein that is trafficked through the ER for ultimate delivery to the plasma membrane. Mutation severely attenuates this processing and a catalytically inactive point mutant, TG1-FLAG(C377A), accumulates in the endoplasmic reticulum and in aggresome-like structures where it is ubiquitinylated. This accumulation results from protein misfolding, as treatment with a chemical chaperone permits it to exit the endoplasmic reticulum and travel to the plasma membrane. ER accumulation is also observed for ichthyosis-associated TG1 mutants. Our findings suggest that misfolding of TG1 mutants leads to ubiquitinylation and accumulation in the ER and aggresomes, and that abnormal intracellular processing of TG1 mutants may be an underlying cause of ichthyosis.