Prospective identification,isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and can give rise to multiple mesenchymal lineages. Because MSCs have only been isolated from tissue in culture, the equivalent cells have not been identified in vivo and little is known about their physiological roles or even their exact tissue location. In this study, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of MSCs (PDGFRα⁺Sca-1⁺CD45⁻TER119⁻) from adult mouse bone marrow. Individual MSCs generated colonies at a high frequency and could differentiate into hematopoietic niche cells, osteoblasts, and adipocytes after in vivo transplantation. Naive MSCs resided in the perivascular region in a quiescent state. This study provides the useful method needed to identify MSCs as defined in vivo entities.Adult BM is composed of hematopoietic stem cells (HSCs) and tissue stem cells, which are often referred to as fibroblast CFUs (CFU-Fs), marrow stromal cells/mesenchymal stem cells (MSCs), or mesenchymal progenitor cells (MPCs; Friedenstein et al., 1974; Prockop, 1997; Conget and Minguell, 1999; Pittenger et al., 1999). As information is gathered about MSCs, parallels are often drawn between them and the extensively characterized HSCs. HSCs were initially identified by Till and McCulloch (1961), who called them spleen CFUs (CFU-Ss), and MSCs were first described by Friedenstein et al. (1974), who called them CFU-Fs. There has since been a major divergence in the way the two stem cell types are studied.HSCs can be identified prospectively by surface markers, isolated by flow cytometry, and transplanted in vivo without being cultured in vitro (Smith et al., 1991; Spangrude et al., 1995; Osawa et al., 1996; Matsuzaki et al., 2004). In contrast, MSCs, which can give rise to multiple mesenchymal cell lineages, including adipocytes, chondrocytes, and osteocytes (Prockop, 1997; Pittenger et al., 1999), are currently isolated by culturing tissues from humans and other species (da Silva Meirelles et al., 2006; Beltrami et al., 2007). Therefore, most information about MSCs comes from in vitro studies (Pittenger et al., 1999) of heterogeneous populations of adherent cells that contain unidentified, putative stem cells. This is a critical difference because it is the ability to isolate HSCs prospectively that has facilitated the rapid progress in understanding their biology. In contrast, because our knowledge of MSCs is based solely on the characterization of cultured cells, it has been virtually impossible to study many of their properties, particularly their function, in vivo.Recent studies consistently show that MSCs not only differentiate into mesenchymal lineage cells but also into neurons (Kohyama et al., 2001; Kondo et al., 2005), skeletal muscle (Dezawa et al., 2005), and myocardium (Makino et al., 1999; Miyahara et al., 2006). Therefore, MSCs are now considered a potentially effective source for stem cell therapy (Jin et al., 2002; Hoffmann et al., 2006). However, safety issues still need to be clarified before their clinical use, particularly because so many biological aspects of MSCs, such as their exact identity and in vivo function, are still unknown.One disadvantage of the conventional in vitro method for isolating MSCs is the unavoidable contamination by hematopoietic cells and the cellular heterogeneity of the cultures, including various fibroblastic cells. In fact, depending on the study, cultured MSCs express a different subset of various cell lineage–specific antigens, adhesion molecules, integrins, and growth factor receptors (Jiang et al., 2002; da Silva Meirelles et al., 2006). Another problem with the current technique is that the cultured cells may acquire different characteristics from their in vivo state, which could include changes in the cell surface markers they express. One example of adherent culture-induced change is seen when MSCs, which are readily expanded in culture without loss of multipotency, show poor tissue tropism when transplanted, including a failure to migrate to the BM (Rombouts and Ploemacher, 2003; Wang et al., 2005; Muguruma et al., 2006; Sackstein et al., 2008), which limits their therapeutic usefulness. In contrast, some studies (including the current one) show that primary BM-derived MSCs (assayed as CFU-Fs) show a low but efficient seeding of the BM upon injection into lethally irradiated hosts (Rombouts and Ploemacher, 2003; Koide et al., 2007). Because these changes affect fundamental properties of the cells, it is difficult to know whether they have retained or lost their original characteristics, including their apparent multipotency, in vitro.Most studies on MSCs have been done using human cells, not murine cells. Murine MSCs (mMSCs) are far more difficult to isolate from the BM and to expand in culture than human MSCs (Phinney et al., 1999; Sun et al., 2003; Peister et al., 2004). Thus, there is much less information on mMSCs than on human MSCs. This reliance on human material means that MSCs cannot be studied in genetic mouse models, which greatly hinders the study of their basic biology, engraftment, and therapeutic potential. In fact, without some means for the prospective isolation and purification of MSCs, it becomes extremely difficult to ascribe any attribute to them with certainty. Hence, there is a clear need for specific markers and methods of detection, enumeration, and isolation of MSCs from the BM and other tissues where they reside.In a previous study, we identified several mesenchymal lineage specific markers in murine BM cells (Koide et al., 2007), and after initial screening, we determined two possible MSC markers (Morikawa et al., 2009), platelet-derived growth factor receptor α (PDGFRα; an early mesodermal marker; Takakura et al., 1997) and stem cell antigen-1 (Sca-1; a known stem cell marker; Ortega et al., 1986).Here, we report a method for identifying and isolating MSCs from the adult murine BM, using flow cytometry in combination with in vitro function assays. We also report our findings on their physiological role, obtained through existing in vivo precursor cell transplantation assays. We describe cell type–specific markers for MSCs, which are useful for their prospective isolation as a highly enriched population that gives rise to mesenchymal cells at the clonal level with high frequency, and demonstrate that these cells are capable of in vivo grafting when transplanted systemically.     

Computational Fluid Dynamics Analysis of Lateral Striate Arteries in Acute Ischemic Stroke Using 7T High-resolution Magnetic Resonance Angiography

Background: Infarcts in the lateral striate artery (LSA) territory can be caused by several pathological changes, including lipohyalinosis and microatheroma. However, fluid dynamic effects on these changes remain unknown. Thus, we investigated whether the fluid dynamic metrics of the LSAs were altered in patients with acute ischemic stroke using computational fluid dynamics (CFD) analysis. Methods: Fifty-one patients with acute ischemic stroke confined in the basal ganglia and/or corona radiata underwent high-resolution magnetic resonance angiography (HR-MRA) at 7T. We performed CFD analyses to obtain indices including the wall shear stress (WSS), WSS gradient (WSSG), and flow velocity (FV) and compared these values between the ipsilesional and contralesional sides in the patients with infarcts in the LSA or non-LSA territories. Results: In patients with LSA-territory infarcts, the WSS, WSSG, and FV values were significantly lower in the ipsilesional LSAs than in the contralesional LSAs (P = .01-.03), while these values in the proximal middle cerebral arteries showed no significant lateralities. In contrast, in patients with non-LSA-territory infarcts, there were no significant lateralities in the metrics between the ipsilesional and contralesional sides. Conclusions: The CFD analyses using HR-MRA revealed significantly low WSS and WSSG values of the ipsilesional LSAs compared with that of the contralesional side in patients with LSA-territory infarcts, suggesting that fluid dynamic factors of LSAs can be one of the risk factors for LSA-territory infarctions.     

Accumulated oropharyngeal water increases coughing during dental treatment with intravenous sedation

Summary  In dental procedures performed under intravenous sedation in patients with intellectual disabilities, procedures are sometimes interrupted by the cough reflex, which may be triggered by intraoral use of water and/or increased secretion stimulating the pharyngeal/laryngeal mucosa, or by those irritating the tracheal mucosa directly through anesthetics-induced impairment of the laryngeal closure reflex. We investigated relationships between frequency of coughing episodes and intraoral use of water, water remaining in the oropharyngeal space, and mean infusion rate of propofol during dental treatments performed under intravenous sedation with midazolam and propofol. Twenty-one intellectually disabled patients were enrolled. After induction, a 14 Fr. suction catheter was inserted nasally, which was fixed where oropharyngeal suction could be done most effectively. Patients were divided into three groups according to the amount of intraoral use of water, amount of oropharyngeal suction and mean infusion rate of propofol. The amount of oropharyngeal suction significantly correlated with intraoral use of water. Frequency of coughing episodes significantly correlated with amount of oropharyngeal suction per minute. However, coughing episodes correlated neither with intraoral use of water nor with infusion rate of propofol. These findings suggested that accumulation of water in the oropharynx increased vulnerability to the cough reflex in dental treatments performed under intravenous sedation.     

Efficacy of S-flurbiprofen plaster in knee osteoarthritis treatment: Results from a phase III,randomized, active-controlled,adequate, and well-controlled trial

Objectives: S-flurbiprofen plaster (SFPP) is a novel non-steroidal anti-inflammatory drug (NSAID) patch, intended for topical treatment for musculoskeletal diseases. This trial was conducted to examine the effectiveness of SFPP using active comparator, flurbiprofen (FP) patch, on knee osteoarthritis (OA) symptoms.

Methods: This was a phase III, multi-center, randomized, adequate, and well-controlled trial, both investigators and patients were blinded to the assigned treatment. Enrolled 633 knee OA patients were treated with either SFPP or FP patch for two weeks. The primary endpoint was improvement in knee pain on rising from the chair as assessed by visual analogue scale (rVAS). Safety was evaluated through adverse events (AEs).

Results: The change in rVAS was 40.9?mm in SFPP group and 30.6?mm in FP patch group (p?p?Conclusions: The superiority of SFPP in efficacy was demonstrated. Most of AEs were mild and few AEs led to treatment discontinuation. Therefore, SFPP provides an additional option for knee OA therapy.     

Repositioning and scleral fixation of subluxated lenses using a T-shaped capsule stabilization hook

We describe a T-shaped capsule stabilization hook (modified capsule expander [M-CE]) used for repositioning and scleral fixation of the lens capsule of subluxated lenses. The 5-0 polypropylene device is flexible and attached to a curved needle. The contact portion is bent at 1.25 mm, and the end bifurcates in a T configuration to form a 3.75 mm footpad from which the capsular bag can be suspended. Modified capsule expanders were implanted in 4 eyes of 4 patients with subluxated cataractous lenses and provided excellent support and centration of the intraocular lens (IOL)-capsular bag complex. The IOLs remained well centered and stable. The corrected distance visual acuity improved to at least 20/20 in all patients after surgery. Thus, M-CEs were effective in fixating the lens capsule to the sclera in patients with significant zonular weakness.     

Dental and craniofacial characteristics in a patient with leprechaunism treated with insulin-like growth factor-I

Leprechaunism is an autosomal recessive disease characterized by elfin-like faces, loss of glucose homeostasis, and severe insulin resistance. This disease is caused by inherited defects of the insulin receptor and is lethal early in life. Perhaps for this reason, the teeth and craniofacial features of patients with leprechaunism have never been reported. In the present case, the patient had been diagnosed with leprechaunism with mutation in the insulin receptor gene and had treatment with recombinant human insulin-like growth factor I (IGF-I) starting at the age of 1 year 7 months. It is of interest that all of his teeth were extremely large and subsequently showed severe crowding in the dental arches. He also showed a large tongue with an anterior open bite. He had a convex facial profile with a remarkably steep mandibular plane angle and large gonial angle. This is the first report of the characteristic phenotypes of the teeth and craniofacial morphology of a patient with leprechaunism treated with IGF-I. In addition, the possible association between these features and long-term IGF-I treatment is discussed.     

An autoregressive linear mixed effects model for the analysis of longitudinal data which include dropouts and show profiles approaching asymptotes

We are interested in longitudinal data of a continuous response that show profiles with an initial sharp change and approaching asymptotes for each patient, and many patients drop out with a reason related to the response. In this paper, we focus on a model that assumes a dropout process is missing at random (MAR). In this dropout process, we can obtain consistent maximum likelihood estimators as long as both the mean and covariance structures are correctly specified. However, parsimonious covariance structures for the profiles approaching asymptotes are unclear. An autoregressive linear mixed effects model can express the profile with random individual asymptotes. We show that this model provides a new parsimonious covariance structure. The covariance structure at steady state is compound symmetry and the other elements of the covariance depend on the measurement points. In simulation studies, the estimate of the asymptote is unbiased in MAR dropouts, but biased in non-ignorable dropouts. We also applied this model to actual schizophrenia trial data.