The Isolation and Manufacture of GMP-Grade Bone Marrow Stromal Cells from Bone Specimens.

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.

Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods.

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

Enabling mesenchymal stromal cell immunomodulatory analysis using scalable platforms.

Human mesenchymal stromal cells (hMSCs) are a promising cell source for numerous regenerative medicine and cell therapy-based applications. However, MSC-based therapies have faced challenges in translation to the clinic, in part due to the lack of sufficient technologies that accurately predict MSC potency and are viable in the context of cell manufacturing. Microfluidic platforms may provide an innovative opportunity to address these challenges by enabling multiparameter analyses of small sample sizes in a high throughput and cost-effective manner, and may provide a more predictive environment in which to analyze hMSC potency. To this end, we demonstrate the feasibility of incorporating 3D culture environments into microfluidic platforms for analysis of hMSC secretory response to inflammatory stimuli and multi-parameter testing using cost-effective and scalable approaches. We first find that the cytokine secretion profile for hMSCs cultured within synthetic poly(ethylene glycol)-based hydrogels is significantly different compared to those cultured on glass substrates, both in growth media and following stimulation with IFN-γ and TNF-α, for cells derived from two donors. For both donors, perfusion with IFN-γ and TNF-α leads to differences in secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and interleukin-1 receptor antagonist (IL-1ra) between hMSCs cultured in hydrogels and those cultured on glass substrates. We then demonstrate the feasibility of analyzing the response of hMSCs to a stable concentration gradient of soluble factors such as inflammatory stimuli for potential future use in potency analyses, minimizing the amount of sample required for dose-response testing.

In vitro co-culture and ex vivo organ culture assessment of primed and cryopreserved stromal cell microcapsules for intervertebral disc regeneration.

Priming towards a discogenic phenotype and subsequent cryopreservation of microencapsulated bone marrow stromal cells (BMSCs) may offer an attractive therapeutic approach for disc repair. It potentially obviates the need for in vivo administration of exogenous growth factors, otherwise required to promote matrix synthesis, in addition to providing 'off-the-shelf' availability. Cryopreserved and primed BMSC microcapsules were evaluated in an in vitro surrogate co-culture model system with nucleus pulposus (NP) cells under intervertebral disc (IVD)-like culture conditions and in an ex vivo bovine organ culture disc model. BMSCs were microencapsulated in alginate microcapsules and primed for 14 d with transforming growth factor beta-3 (TGF-ß3) under low oxygen conditions prior to cryopreservation. For the in vitro phase, BMSC microcapsules (unprimed or primed) were cultured for 28 d in a surrogate co-culture model system mimicking that of the IVD. For the ex vivo phase, microcapsules (unprimed or primed) were injected into the NP of bovine discs that underwent nucleotomy. In vitro results revealed that although NP cells produced significantly more matrix components in co-culture with BMSC microcapsules regardless of the differentiation state, unprimed microcapsules were inadequate at synthesising matrix as compared to primed microcapsules. However, this difference was diminished when evaluated in the ex vivo organ culture model,withboth unprimed and primed BMSC microcapsules accumulating large amounts of sulphated glycosaminoglycan (sGAG) and collagen and filling the defect cavity. Both models demonstrated that cryopreservation of BMSC microcapsules may offer a feasible strategy for predesigned delivery through cryobanking for on-demand regeneration of the IVD.

Comparison of intraoperative procedures for isolation of clinical grade stromal vascular fraction for regenerative purposes: a systematic review.

Intraoperative application of the stromal vascular fraction (SVF) of adipose tissue requires a fast and efficient isolation procedure of adipose tissue. This review was performed to systematically assess and compare procedures currently used for the intraoperative isolation of cellular SVF (cSVF) and tissue SVF (tSVF) that still contain the extracellular matrix. Pubmed, EMBASE and the Cochrane central register of controlled trials databases were searched for studies that compare procedures for intraoperative isolation of SVF (searched 28 September 2016). Outcomes of interest were cell yield, viability of cells, composition of SVF, duration, cost and procedure characteristics. Procedures were subdivided into procedures resulting in a cSVF or tSVF. Thirteen out of 3038 studies, evaluating 18 intraoperative isolation procedures, were considered eligible. In general, cSVF and tSVF intraoperative isolation procedures had similar cell yield, cell viability and SVF composition compared to a nonintraoperative (i.e. culture laboratory-based collagenase protocol) control group within the same studies. The majority of intraoperative isolation procedures are less time consuming than nonintraoperative control groups, however. Intraoperative isolation procedures are less time-consuming than nonintraoperative control groups with similar cell yield, viability of cells and composition of SVF, and therefore more suitable for use in the clinic. Nevertheless, none of the intraoperative isolation procedures could be designated as the preferred procedure to isolate SVF. Copyright © 2017 John Wiley & Sons, Ltd.

Non-linear optical microscopy as a novel quantitative and label-free imaging modality to improve the assessment of tissue-engineered cartilage.

OBJECTIVE: Current systems to evaluate outcomes from tissue-engineered cartilage (TEC) are sub-optimal. The main purpose of our study was to demonstrate the use of second harmonic generation (SHG) microscopy as a novel quantitative approach to assess collagen deposition in laboratory made cartilage constructs. METHODS: Scaffold-free cartilage constructs were obtained by condensation of in vitro expanded Hoffa's fat pad derived stromal cells (HFPSCs), incubated in the presence or absence of chondrogenic growth factors (GF) during a period of 21 d. Cartilage-like features in constructs were assessed by Alcian blue staining, transmission electron microscopy (TEM), SHG and two-photon excited fluorescence microscopy. A new scoring system, using second harmonic generation microscopy (SHGM) index for collagen density and distribution, was adapted to the existing "Bern score" in order to evaluate in vitro TEC. RESULTS: Spheroids with GF gave a relative high Bern score value due to appropriate cell morphology, cell density, tissue-like features and proteoglycan content, whereas spheroids without GF did not. However, both TEM and SHGM revealed striking differences between the collagen framework in the spheroids and native cartilage. Spheroids required a four-fold increase in laser power to visualize the collagen matrix by SHGM compared to native cartilage. Additionally, collagen distribution, determined as the area of tissue generating SHG signal, was higher in spheroids with GF than without GF, but lower than in native cartilage. CONCLUSION: SHG represents a reliable quantitative approach to assess collagen deposition in laboratory engineered cartilage, and may be applied to improve currently established scoring systems.

hCG activates Epac-Erk1/2 signaling regulating Progesterone Receptor expression and function in human endometrial stromal cells.

STUDY QUESTION: How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? SUMMARY ANSWER: hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. WHAT IS KNOWN ALREADY: hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. STUDY DESIGN, SIZE, DURATION: This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. WIDER IMPLICATIONS OF THE FINDINGS: We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.

Functional assessment of hematopoietic niche cells derived from human embryonic stem cells.

To evaluate hematopoietic niche cell populations isolated from human embryonic stem cells (hESCs), we tested the ability of hESC-derived stromal lines to support CD34(+) umbilical cord blood (UCB)- and hESC-derived CD34(+)45(+) cells in long-term culture initiating cell (LTC-IC) assays. Specifically, these hematopoietic populations were cocultured with hESC-derived mesenchymal stromal cells (hESC-MSCs) and hESC-derived endothelial cells (hESC-ECs), and then assessed for their LTC-IC potential in comparison to coculture with bone marrow (BM)-derived MSCs and the mouse stromal line M2-10B4. We found that the hESC-derived stromal lines supported LTC-ICs from UCB similar to M2-10B4 cells and better than BM-MSCs. However, none of the stromal populations supported LTC-IC from hESC-derived CD34(+)45(+) cells. Engraftment data using the output from LTC-IC assays showed long-term repopulation (12 weeks) of NSG mice to correlate with LTC-IC support on a given stromal layer. Therefore, hESC-derived stromal lines can be used to efficiently evaluate putative hematopoietic stem/progenitor cells derived from hESCs or other cell sources.

Cytompatibility assessment of the surface of titanium after phosphorylation.

In this study, orthophosphoric acid (H3PO4) in the treatment of porous titanium (Ti) is investigated and the ability of rat bone marrow stromal cells (BMSCs) is assessed to proliferate and differentiate on these modified surfaces in vitro. To improve the cytocompatibility of Ti surfaces, pure Ti was activated commercially by simple chemical pretreatment in H3PO4 with different densities. Next, the phosphorylated specimens were soaked in simulated body fluid (SBF) to study the effect of biomineralization. The3-[4,5-dimethylthiazol-2-y1]-2, 5-diphenytetrazolium bromide (MTT) assay and the measurement of alkaline phosphatase (ALP) activity utilized to assess proliferation and differentiation of BMSCs on exposure to modified Ti surfaces. Scanning electron microscopic (SEM) images showed that the surfaces of the pre-treated samples were characterized by a complex structure which consisted of a mesh-like morphological matrix and an uniform surface with different morphic crystals of titanium dihydrogen orthophosphate (Ti(H2PO4)3). These crystals contained hydroxyl with phosphate residues that resulted in biomineralization of cells. Therefore, BMSCs reveales a well-dispersed morphology on these modified and functionalized Ti surfaces. The viability and ALP activity of BMSCs on these altered biomimetic surfaces are found to be greater than those of the controls. It is concluded that the treatment of Ti by acid etching in orthophosphoric acid is a suitable method to enhance the in vitro proliferation and differentiation of BMSCs.

The assessment of cryopreservation conditions for human umbilical cord stroma-derived mesenchymal stem cells towards a potential use for stem cell banking.

Human umbilical cord stroma-derived mesenchymal stem cells (hUCS-MSCs) are considered as a remarkable and promising stem cell source to be potentially used in cellular therapies. While no graft rejection has been reported in the recipient organism even in xeno-transplantation studies, attenuate tumor cell growth and gene transfers have been experimentally shown. In this study, we have demonstrated a reliable, reproducible and efficient cryopreservation method of hUCS-MSCs resulting in one of the highest cell survival rates reported so far. Conventional, computer-controlled multistep slow freezing (MSSF), and vitrification methods were comparatively tested using cell permeable [dimethylsulfoxide (DMSO), ethylene glycol] and impermeable [trehalose, sucrose, hydroxyethyl starch (HES), human serum albumin] cryoprotectant agents (CPAs). After determining the ice nucleation point for each solution, latent heat evolution was suppressed during freezing, followed by a cooling process to -40°C at 1°C/min or 0.3°C/min. The efficiency of the cryopreservation techniques used was determined by cell viability and proliferation assays, the expression of cell surface markers, cytoskeletal proteins and chromosome alignments. The cell survival rate was found to be highest (87 ± 5%) by MSSF with sucrose (0.1 M) +DMSO (10%) at 1°C/min freezing rate. In this group, no significant difference was noted before and after the cryopreservation in cell morphology, cytokeratin, vimentin, and α-smooth muscle actin profiles and the expressions of CD105, CD90, CD73, CD29 and HLA-DR. Second highest cell survival ratio (85 ± 6%) was obtained in DMSO (10%) alone at 1°C/min freezing rate. Interestingly, poor (18 ± 15%) cell survival rates were obtained after vitrification. Cumulatively, results indicated that MSSF favors the other freezing protocols with an addition of sucrose or DMSO alone depending on the freezing rate used.

Long-term in-vivo tumorigenic assessment of human culture-expanded adipose stromal/stem cells.

After more than a decade of extensive experimentation, the promise of stem cells to revolutionize the field of medicine has negotiated their entry into clinical trial. Adipose tissue specifically holds potential as an attainable and abundant source of stem cells. Currently undergoing investigation are adipose stem cell (ASC) therapies for diabetes and critical limb ischemia, among others. In the enthusiastic pursuit of regenerative therapies, however, questions remain regarding ASC persistence and migration, and, importantly, their safety and potential for neoplasia. To date, assays of in vivo ASC activity have been limited by early end points. We hypothesized that with time, ASCs injected subcutaneously undergo removal by normal tissue turnover and homeostasis, and by the host's immune system. In this study, a high dose of culture expanded ASCs was formulated and implanted as multicellular aggregates into immunocompromised mice, which were maintained for over one year. Animals were monitored for toxicity, and surviving cells quantified at study endpoint. No difference in growth/weight or lifespan was found between cell-treated and vehicle treated animals, and no malignancies were detected in treated animals. Moreover, real-time PCR for a human specific sequence, ERV-3, detected no persistent ASCs. With the advent of clinical application, clarification of currently enigmatic stem cell properties has become imperative. Our study represents the longest duration determination of stem cell activity in vivo, and contributes strong evidence in support of the safety of adipose derived stem cell applications.

Comparative assessment of the osteoconductive properties of different biomaterials in vivo seeded with human or ovine mesenchymal stem/stromal cells.

Mesenchymal stromal/stem cells (MSC), when used in combination with biomaterial scaffolds, have been shown to contribute at varying efficiencies to bone and cartilage regeneration in preclinical large animal models and human clinical trials. In an orthopedic context, identification of the optimal scaffold, which is capable of inducing tissue regeneration, has been the subject of numerous studies. In the present study, we show that ex vivo-expanded MSC from human and ovine bone marrow display similar phenotypic properties, but exhibit differences in their ability to form bone in vivo when transplanted with different biocompatible scaffold composites. We found that the ovine MSC formed ectopic bone on all scaffolds tested with the exception of collagen-based demineralized bone matrix. In contrast, human MSC in general formed less bone and only on those biomaterials composed of ceramic particles containing at least 15% hydroxyapatite. This study demonstrates the differences in bone formation potential between human and ovine MSC in vivo based on the osteoconductive properties of different bioscaffolds currently being used for orthopedic clinical applications.

Assessment of presence and characteristics of multipotent stromal cells in human endometrium and decidua.

This review discusses the presence and characteristics of multipotent stromal cells in human endometrium and decidua. A number of research groups have reported the isolation and characterization of multipotent stromal cells from the basal layer of the endometrium, and in a single case just from the menstrual blood, i.e. the superficial functional layer. Similarly, multipotent pre-decidual stromal cells are isolated from early decidua and characterized accordingly. Multipotent endometrial stromal cells and multipotent decidual stromal cells are shown to express the basic features of adult stem cells, which are clonogenicity, self-renewal, a potential to differentiate into adipogenic, osteogenic, chrondrogenic, endothelial-like cells and a specific set of surface molecules (CD73, CD90 and CD105). So far, it is not clear whether the same population of multipotent stromal cells is isolated from the basal endometrium or early decidua because it has been shown that in some cases the differentiation potential of endometrial stromal cells is more restricted in comparison to the decidual stromal cells. It is reasonable to assume that it is one cell population under different control by hormonal, paracrine and autocrine factors. Thus far, the functions of these cells have not been convincingly revealed.

Macroporous hydroxyapatite scaffolds for bone tissue engineering applications: physicochemical characterization and assessment of rat bone marrow stromal cell viability.

In this work, a new methodology is reported for developing hydroxyapatite (HA) scaffolds using an organic sacrifice template. The novelty of work consists of possibility of obtaining porous and highly interconnected scaffolds mimicking the sacrificial component. Our purpose consisted of evaluating the physicochemical properties of the HA scaffolds by means of Fourier transform infra-red spectroscopy, X-ray diffraction analysis, and scanning electron microscopy (SEM) attached with an X-ray detector. The HA scaffolds obtained possess a porosity of approximately 70%, and macropores diameter in the range of 50-600 microm. In contrast, results regarding the microcomputed tomography analysis have demonstrated both high pore uniformity and interconnectivity across the scaffolds. The compressive strength of the HA scaffolds was found to be 30.2 +/- 6.0 MPa. Bioactivity of the HA scaffolds was assessed by immersion into a simulated body fluid solution, in vitro. SEM observations have showed a deposition of apatite on the surface of the HA scaffolds, with a "cauliflower-like" morphology after 1 day, and tend to be more pronounced with the immersion time. The changes in calcium and phosphorus concentration were monitored by inductively-coupled plasma optical emission spectrometry. Cytotoxicity of the HA scaffolds was preliminarily investigated by carrying direct observation of mouse fibroblasts cells (L929 cell-line) death in the inverted microscope, and then cell viability was determined by means of carrying out a MTS assay. Complementarily, a luminescent cell viability assay based on the quantification of adenosine triphosphate was performed using rat bone marrow stromal cells (RBMSCs). A LIVE/DEAD assay and SEM analysis allowed the visualization of the RBMSCs adhesion and proliferation on the surface of the HA scaffolds. According to the results obtained from 3D architecture, mechanical properties, biocompatibility, and adhesion tests, it is suggested that HA scaffolds has potential to find applications in bone tissue engineering scaffolding.

Assessment of VEGF-receptor system expression in the porcine endometrial stromal cells in response to insulin-like growth factor-I, relaxin, oxytocin and prostaglandin E2.

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.

Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells.

Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by beta-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect. Disclosure of potential conflicts of interest is found at the end of this article.

Mechanical loading down-regulates peroxisome proliferator-activated receptor gamma in bone marrow stromal cells and favors osteoblastogenesis at the expense of adipogenesis.

Because a lack of mechanical information favors the development of adipocytes at the expense of osteoblasts, we hypothesized that the peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent balance between osteoblasts and adipocytes is affected by mechanical stimuli. We tested the robustness of this hypothesis in in vivo rodent osteogenic exercise, in vitro cyclic loading of cancellous haversian bone samples, and cyclic stretching of primary stromal and C3H10T1/2 cells. We found that running rats exhibit a decreased marrow fat volume associated with an increased bone formation, presumably through recruitment of osteoprogenitors. In the tissue culture model and primary stromal cells, cyclic loading induced higher Runx2 and lower PPARgamma2 protein levels. Given the proadipocytic and antiosteoblastic activities of PPARgamma, we studied the effects of cyclic stretching in C3H10T1/2 cells, treated either with the PPARgamma activator, Rosiglitazone, or with GW9662, a potent antagonist of PPARgamma. We found, through both cytochemistry and analysis of lineage marker expression, that under Roziglitazone cyclic stretch partially overcomes the induction of adipogenesis and is still able to favor osteoblast differentiation. Conversely, cyclic stretch has additive effects with GW9662 in inducing osteoblastogenesis. In conclusion, we provide evidence that mechanical stimuli are potential PPARgamma modulators counteracting adipocyte differentiation and inhibition of osteoblastogenesis.

MR assessment of osteogenic differentiation in tissue-engineered constructs.

Bone marrow stromal cells (MSC) are a promising source of osteoprogenitor cells for bone tissue engineering. However, the population of the osteoprogenitor cells and their differentiation potentials change with the gender, age, and health of the donor. Development of a noninvasive method to assess osteogenic progression is critical for successful bone tissue regeneration. High-resolution magnetic resonance imaging (MRI) (at 11.7 T, with spatial resolution of 62.5 x 62.5 microm in 500 microm slices) is used in the present study to monitor osteogenic differentiation of tissue-engineered constructs prepared by seeding human bone MSCs on gelatin sponge scaffolds. Quantitative measurements of the MR relaxation times (T1, T2) and the apparent diffusion coefficient (ADC) were performed for four successive weeks on control tissue constructs and constructs exposed to osteogenic differentiation medium. The T1 and T2 relaxation times and ADC were found to decrease as osteogenic progression proceeded in samples exposed to osteogenic differentiation medium. At week 4, the T1, T2, and ADC of TE constructs were 1.81 +/- 0.11 s, 19.5 +/- 11.02 ms, and 1.01 +/- 0.47 x 10(3) mm(2)/s, respectively, for osteogenic differentiated constructs, significantly different from control constructs 2.22 +/- 0.08 s, 50.39 +/- 5.57 ms, and 1.86 +/- 0.18 x 107(3) mm(2)/s (p < 0.05). The MR parameters were also highly correlated with the cell seeding densities and alkaline phosphatase (ALP) activities of the osteogenic constructs. In conclusion, periodic measurements of MR parameters (T1, T2, and ADC) provide a promising method for noninvasive monitoring of the status of tissue-engineered bone growth and differentiation.

Endometrial cells in cervical cytology: review of cytological features and clinical assessment.

The 2001 Bethesda System for Reporting Cervical Cytology recommends reporting benign exfoliated endometrial cells in women age 40 and older, and a review of the literature supports this recommendation. Stromal cells and histiocytes do not need to be reported. The effect of hormonal therapy on endometrial shedding is reviewed. Clinical information should be provided to the laboratory so that appropriate educational notes can be appended to the cytology report. Benign endometrial cells in premenopausal women in the first half of the cycle are not associated with significant pathology and such women do not need additional evaluation. Significant pathology is also unlikely in the second half of the cycle and evaluation may not be required unless clinically indicated. Initial evaluation of other women with benign endometrial cells may include either endometrial sampling or transvaginal ultrasound. Atypical endometrial cells are associated with a higher rate of significant pathology and should lead to additional evaluation. Additional prospective studies on the management of patients with endometrial cells on Pap tests are needed.

A pre-clinical assessment model of rat autogeneic bone marrow stromal cell transplantation into the central nervous system.

In order to verify the biological aspects of 'autogeneic' bone marrow stromal cells (BMSC) transplantation for neurological disorders, we aimed our study towards the assessment of the survival, distribution, and differentiation of autologous BMSC in the central nervous system (CNS). We harvested rat BMSC from femur bones, and the nuclei were then fluorescently labeled by a 24-h co-culture with bis-benzimide. These BMSC were stereotactically injected into the striatum (n=6) or thoracic cord (n=8) of each animal. We evaluated the distribution and differentiation of 'autogeneic' BMSC in the brain and spinal cord after 4 weeks, using the immunohistochemistry technique. We found some injected cells in the ipsilateral striatum, hippocampus, neocortex, and bilateral corpus callosum, and approximately 20% and 15% of the engrafted cells expressed neuronal and astrocytic markers, respectively. Other injected cells were distributed in the dorsal funiculus and adjacent gray matter, and about 10% and 15% of these cells expressed neuronal and astrocytic markers, respectively. Although the precise mechanism of BMSC transdifferentiation still remains unclear, the present results show that 'autogeneic' BMSC could highly differentiate into their own CNS neural cells, suggesting that they are surrounded by favorable conditions.