Tracking mesenchymal stromal cells using an ultra‐bright TAT‐functionalized plasmonic‐active nanoplatform

High‐resolution tracking of stem cells remains a challenging task. An ultra‐bright contrast agent with extended intracellular retention is suitable for in vivo high‐resolution tracking of stem cells following the implantation. Here, a plasmonic‐active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide‐functionalized gold nanostars (TAT‐GNS) that emit ultra‐bright two‐photon photoluminescence capable of tracking MSCs under high‐resolution optical imaging. In vitro experiment showed TAT‐GNS‐labeled MSCs retained a similar differentiability to that of non‐labeled MSCs controls. Due to their star shape, TAT‐GNS exhibited greater intracellular retention than that of commercial Q‐Tracker. In vivo imaging of TAT‐GNS‐labeled MSCs five days following intra‐arterial injections in mice kidneys showed possible MSCs implantation in juxta‐glomerular (JG) regions, but non‐specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic‐active nanoplatforms may be a useful intracellular tracking tool for stem cell research.

An ultra‐bright intracellular contrast agent is developed using TAT peptide‐functionalized gold nanostars (TAT‐GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two‐photon photoluminescence and superior labeling efficiency than commercial Q‐Tracker. Following renal implantation, some TAT‐GNS‐labeled MSCs permeate blood vessels and migrate to the juxta‐glomerular region.     

Application of cell penetrating peptide in magnetic resonance imaging of bone marrow mesenchymal stem cells

Tracking the distribution and differentiation of stem cells by high-resolution imaging techniqueswould have significant clinical and research implications.In this study,a model cell-penetrating peptide wasused to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs).MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro.The cell-penetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by asolid-phase peptide synthesis method.Fluorescein imaging analysis confirmed that this new peptide couldinternalize into the cytoplasm and nucleus at room temperature,4℃ and 37℃.Gadolinium were efficientlyinternalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner,resulting in intercellular shortening of longitudinal relaxation enhancements,which were obviously detectedby 1.5 Tesla Magnetic Resonance Imaging.Cytotoxicity assay and flow cytometric analysis showed thatthe intercellular contrast medium incorporation did not affect cell viability at the tested concentrations.Thein vitro experiment results suggested that the new constructed peptides could be a vector for trackingMSCs.     

Photoacoustic Monitoring of the Viability of Mesenchymal Stem Cells Labeled with Indocyanine Green

Background

Stem cell therapy has a huge potential to enhance the recovery of damaged tissues and organs. However, it has been reported that majority of implanted stem cells cannot survive after implantation. Therefore, noninvasive monitoring of stem cell viability is essential to estimate the efficacy of stem cell therapy. However, current imaging methods have disadvantages for monitoring of stem cell viability such as cost, penetration depth, and safety. To overcome the limitations, photoacoustic imaging well known for sufficient penetration depth, relatively low cost, and non-ionizing radiation can be a novel alternative assessment method of stem cell viability.

间充质干细胞双标记光学成像的体内试验研究

目的:验证双标记生物发光成像活体观测MSCs在肝癌裸鼠模型向肿瘤病灶的趋化作用的可行性。方法:应用fluorescence(荧光)与bioluminescence(生物发光)两种成像方法,对MSCs进行CM-Di I荧光标记及对人肝癌细胞Hep G2进行Fluc-慢病毒感染并由此建立裸鼠肝癌模型,构建双标记成像系统,应用精诺真小动物光学成像仪在裸鼠肝癌模型中观测间充质干细胞向肿瘤的趋化作用。结果:在鼠尾静脉注射标记MSCs细胞后21天荧光成像可见MSCs主要积聚于肿瘤病灶处及肝脏。生物发光成像后可监测到病灶处由luciferase标记肿瘤细胞(Hep G2)发出荧光;将荧光成像与生物发光成像所得图像经后处理融合后,可见证间充质干细胞像肿瘤病灶定向迁徙的生物过程。经肿瘤病理切片证实间充质干细胞成功迁徙至肿瘤病灶中。结论:应用间充质干细胞双标记光学成像系统实现MSCs在活体内对肿瘤的趋化过程进行观测是可行的。这种成像方法可作为下一步以MSCs为载体的肿瘤基因治疗的有效监测手段。     

Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration

Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.     

Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells.

Magnetic resonance (MR) tracking of magnetically labeled stem and progenitor cells is an emerging technology, leading to an urgent need for magnetic probes that can make cells highly magnetic during their normal expansion in culture. We have developed magnetodendrimers as a versatile class of magnetic tags that can efficiently label mammalian cells, including human neural stem cells (NSCs) and mesenchymal stem cells (MSCs), through a nonspecific membrane adsorption process with subsequent intracellular (non-nuclear) localization in endosomes. The superparamagnetic iron oxide nanocomposites have been optimized to exhibit superior magnetic properties and to induce sufficient MR cell contrast at incubated doses as low as 1 microg iron/ml culture medium. When containing between 9 and 14 pg iron/cell, labeled cells exhibit an ex vivo nuclear magnetic resonance (NMR) relaxation rate (1/T2) as high as 24-39 s-1/mM iron. Labeled cells are unaffected in their viability and proliferating capacity, and labeled human NSCs differentiate normally into neurons. Furthermore, we show here that NSC-derived (and LacZ-transfected), magnetically labeled oligodendroglial progenitors can be readily detected in vivo at least as long as six weeks after transplantation, with an excellent correlation between the obtained MR contrast and staining for beta-galactosidase expression. The availability of magnetodendrimers opens up the possibility of MR tracking of a wide variety of (stem) cell transplants.     

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.     

In vitro labeling of human umbilical cord mesenchymal stem cells with superparamagnetic iron oxide nanoparticles

Human umbilical cord mesenchymal stem cells (hUC‐MSCs) transplantation has been shown to promote regeneration and neuroprotection in central nervous system (CNS) injuries and neurodegenerative diseases. To develop this approach into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Neural precursor cells and hematopoietic stem cells can be efficiently labeled by superparamagnetic iron oxide (SPIO) nanoparticle. The purpose of our study was to prospectively evaluate the influence of SPIO on hUC‐MSCs and the feasibility of tracking for hUC‐MSCs by noninvasive imaging. In vitro studies demonstrated that magnetic resonance imaging (MRI) can efficiently detect low numbers of SPIO‐labeled hUC‐MSCs and that the intensity of the signal was proportional to the number of labeled cells. After transplantation into focal areas in adult rat spinal cord transplanted SPIO‐labeled hUC‐MSCs produced a hypointense signal using T2‐weighted MRI in rats that persisted for up to 2 weeks. This study demonstrated the feasibility of noninvasive imaging of transplanted hUC‐MSCs. J. Cell. Biochem. 108: 529–535, 2009. © 2009 Wiley‐Liss, Inc.     

Magnetic resonance imaging of ferumoxide-labeled mesenchymal stem cells in cartilage defects: in vitro and in vivo investigations

The purpose of this study was to (1) compare three different techniques for ferumoxide labeling of mesenchymal stem cells (MSCs), (2) evaluate if ferumoxide labeling allows in vivo tracking of matrix-associated stem cell implants (MASIs) in an animal model, and (3) compare the magnetic resonance imaging (MRI) characteristics of ferumoxide-labeled viable and apoptotic MSCs. MSCs labeled with ferumoxide by simple incubation, protamine transfection, or Lipofectin transfection were evaluated with MRI and histopathology. Ferumoxide-labeled and unlabeled viable and apoptotic MSCs in osteochondral defects of rat knee joints were evaluated over 12 weeks with MRI. Signal to noise ratios (SNRs) of viable and apoptotic labeled MASIs were tested for significant differences using t-tests. A simple incubation labeling protocol demonstrated the best compromise between significant magnetic resonance signal effects and preserved cell viability and potential for immediate clinical translation. Labeled viable and apoptotic MASIs did not show significant differences in SNR. Labeled viable but not apoptotic MSCs demonstrated an increasing area of T2 signal loss over time, which correlated to stem cell proliferation at the transplantation site. Histopathology confirmed successful engraftment of viable MSCs. The engraftment of iron oxide-labeled MASIs by simple incubation can be monitored over several weeks with MRI. Viable and apoptotic MASIs can be distinguished via imaging signs of cell proliferation at the transplantation site.     

菲立磁标记食蟹猴骨髓间充质干细胞的脑内移植示踪研究

中枢神经系统损伤后的再生修复问题一直是神经科学领域关注的重点之一,骨髓间充质干细胞移植治疗拓宽了人类中枢神经系统损伤的治疗前景,而非侵入性的磁共振成像能活体追踪移植细胞,评价移植效果。应用菲立磁标记食蟹猴骨髓来源的间充质干细胞,在脑立体定位仪引导下,自体脑内移植。结果显示,菲立磁标记间充质干细胞的有效率高达90%以上,移植区磁共振影像呈明显的低信号改变。标记的间充质干细胞移植后在脑内存活,并向周围的脑实质内迁移。移植8周后,发现移植细胞通过血管向对侧脑部迁移,但并未发现移植细胞向神经细胞分化。这些结果提示,菲立磁可用于标记、追踪脑内移植的食蟹猴骨髓间充质干细胞,标记的移植细胞可在脑内存活、迁移。     

A new methodology of mesenchymal stem cell expansion using magnetic nanoparticles

Mesenchymal stem cells (MSCs), which can differentiate into multiple mesodermal tissues, may be useful for autologous cell transplantation, if MSCs, which are isolated from bone marrow in small numbers, can be expanded in vitro. We developed a combined methodological approach to enrich and proliferate MSCs in vitro using magnetic nanoparticles. Our magnetite cationic liposomes (MCLs), which have a positive surface charge in order to improve adsorption, accumulated in MSCs at a concentration of 20 pg of magnetite per cell. The MCLs exhibited no toxicity against MSCs in proliferation and differentiation to osteoblasts and adipocytes. The MSCs magnetically labeled by MCLs were enriched using magnets and then cultured, resulting in much higher density (seeding density, 1000 cells/cm²) than in ordinary culture (seeding density, 18 cells/cm²). When MSCs were seeded at high density using MCLs, there was a 5-fold increase in the number of cells, compared to culture prepared without MCLs. Our results suggest that this novel culture method using magnetic nanoparticles can be used to efficiently expand MSCs for clinical application.     

Migration, fate and in vivo imaging of adult stem cells in the CNS

Adult stem cells have been intensively studied for their potential use in cell therapies for neurodegenerative diseases, ischemia and traumatic injuries. One of the most promising cell sources for autologous cell transplantation is bone marrow, containing a heterogenous cell population that can be roughly divided into hematopoietic stem and progenitor cells and mesenchymal stem cells (MSCs). MSCs are multipotent progenitor cells that, in the case of severe tissue ischemia or damage, can be attracted to the lesion site, where they can secrete bioactive molecules, either naturally or through genetic engineering. They can also serve as vehicles for delivering therapeutic agents. Mobilized from the marrow, sorted or expanded in culture, MSCs can be delivered to the damaged site by direct or systemic application. In addition, MSCs can be labeled with superparamagnetic nanoparticles that allow in vivo cell imaging. Magnetic resonance imaging (MRI) is thus a suitable method for in vivo cell tracking of transplanted cells in the host organism. This review will focus on cell labeling for MRI and the use of MSCs in experimental and clinical studies for the treatment of brain and spinal cord injuries.     

Cell‐assembled nanoclusters of MSC‐targeting Gd‐DOTA‐peptide as a T2 contrast agent for MRI cell tracking

A cyclic peptide CC9 that targets cell membrane of mesenchymal stem cells (MSCs) is coupled with Gd‐DOTA to yield a Gd‐DOTA‐CC9 complex as MRI contrast agent. It is used to label human MSCs (hMSCs) via electroporation. Electroporation‐labeling of hMSCs with Gd‐DOTA‐CC9 induces cell‐assembly of Gd‐DOTA‐CC9 nanoclusters in the cytoplasm, significantly promotes cell‐labeling efficacy and intracellular retention time of the agent. In vitro MRI of labeled hMSCs exhibits significant signal reduction under T₂‐weighted MRI, which can allow long‐term tracking of labeled cell transplants in in vivo migration. The labeling strategy is safe in cytotoxicity and differentiation potential.     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

The application of super paramagnetic iron oxide-labeled mesenchymal stem cells in cell-based therapy

Mesenchymal stem cell (MSC)-based therapy has great potential for tissue regeneration. However, being able to monitor the in vivo behavior of implanted MSCs and understand the fate of these cells is necessary for further development of successful therapies and requires an effective, non-invasive and non-toxic technique for cell tracking. Super paramagnetic iron oxide (SPIO) is an idea label and tracer of MSCs. MRI can be used to follow SPIO-labeled MSCs and has been proposed as a gold standard for monitoring the in vivo biodistribution and migration of implanted SPIO-labeled MSCs. This review discusses the biological effects of SPIO labeling on MSCs and the therapeutic applications of local or systemic delivery of these labeled cells.     

Telomerase deficiency impairs differentiation of mesenchymal stem cells

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.     

Bone marrow mesenchymal stem cells suppress lymphocyte proliferation in vitro but fail to prevent graft-versus-host disease in mice

Several reports have suggested that mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect in vitro, and thus may have a therapeutic potential for T cell-dependent pathologies. We aimed to establish whether MSCs could be used to control graft-vs-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation. From C57BL/6 and BALB/c mouse bone marrow cells, we purified and expanded MSCs characterized by the lack of expression of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells. These MSCs suppressed alloantigen-induced T cell proliferation in vitro in a dose-dependent manner, independently of their MHC haplotype. However, when MSCs were added to a bone marrow transplant at a MSCs:T cells ratio that provided a strong inhibition of the allogeneic responses in vitro, they yielded no clinical benefit on the incidence or severity of GVHD. The absence of clinical effect was not due to MSC rejection because they still could be detected in grafted animals, but rather to an absence of suppressive effect on donor T cell division in vivo. Thus, in these murine models, experimental data do not support a significant immunosuppressive effect of MSCs in vivo for the treatment of GVHD.     

Comparative immunomodulatory properties of adipose‐derived mesenchymal stem cells conditioned media from BALB/c,C57BL/6, and DBA mouse strains

Adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) have been shown to be capable of differentiating into multiple cell type and exert immunomodulatory effects. Since the selection of ideal stem cell is apparently crucial for the outcome of experimental stem cell therapies, therefore, in this study we compared AD‐MSCs conditioned media (CM) from BALB/c, C57BL/6, and DBA mouse strains. No significant difference was found in the morphology, cell surface markers, in vitro differentiation and proliferation potentials of AD‐MSCs isolated from C57BL/6, BALB/c, and DBA mice. The immunological assays showed some variation among the strains in the cytokines, nitric oxide (NO), and indoleamine 2,3‐dioxygenase (IDO) production and immunomodulatory effects on splenocytes functions. Our results indicated a suppression of splenocytes proliferation in the presence of AD‐MSC CM from the three inbred mouse strains. However, BALB/c CM exerted a higher suppression of splenocytes proliferation. AD‐MSCs isolated from C57BL/6 and BALB/c mice produced higher levels of TGF‐β than those from DBA mice. Furthermore, IL‐17 and IDO production was higher in AD‐MSCs isolated from BALB/c mice. Our results indicated an increased production of TGF‐β, IL‐4, IL‐10, NO, and IDO by splenocytes in response to CM from BALB/c AD‐MSCs. In conclusion, our results showed that the immunomodulatory properties of mouse AD‐MSCs is strain‐dependent and this variation should be considered during selection of appropriate stem cell source for in vivo experiments and stem cell therapy strategies. J. Cell. Biochem. 114: 955–965, 2013. © 2012 Wiley Periodicals, Inc.     

Enhanced development of porcine embryos cloned from bone marrow mesenchymal stem cells

In the present study, we have characterized an isolated population of porcine bone marrow mesenchymal stem cells (MSCs) for multilineage commitment and compared the developmental potential of cloned embryos with porcine MSCs and fetal fibroblasts (FFs). MSCs exhibited robust alkaline phosphatase activity and later transformed into mineralized nodules following osteoinduction. Furthermore, MSCs underwent adipogenic and chondrogenic differentiation by producing lipid droplets and proteoglycans, respectively. Primary cultures of FFs from a female fetus at ~30 day of gestation were established. Donor cells at 3-4 passage were employed for nuclear transfer (NT). Cell cycle analysis showed that the majority of MSCs in confluence were in the G0/G1 stage. Cumulus-oocyte complexes were matured and fertilized in vitro (IVF) as control. The cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs and FFs (84.54.6% vs. 52.25.4% and 50.85.2%, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs (20.62.5% and 18.43.0%) did not differ, but were significantly (P<0.05) higher than NT derived from FFs (9.52.1%). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs (34.45.2 and 0.380.08, respectively) were significantly (P<0.05) higher than those from FFs (22.65.5 and 0.180.12, respectively). Proportions of TUNEL positive cells in NT embryos from FFs (7.31.8%) were significantly (P<0.05) higher than in MSCs (4.61.3%) and IVF (2.50.9%). The results clearly demonstrate that multipotent bone marrow MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned porcine embryos.