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Acute and chronic solid organ failures are costly disease processes with high mortality rates. Inflammation plays a central role in both acute and chronic organ failure, including heart, lung and kidney. In this regard, new therapies for these disorders have focused on inhibiting the mediators of inflammation, including cytokines and free radicals, with little or no success in clinical studies. Recent novel treatment strategies have been directed to cell-based rather than mediator-based approaches, designed to immunomodulate the deleterious effects of inflammation on organ function. One approach, cell therapy, replaces cells that were damaged in the acute or chronic disease process with stem/progenitor technology, to rebalance excessive inflammatory states. As an example of this approach, the use of an immunomodulatory role of renal epithelial progenitor cells to treat acute renal failure (ARF) and multiorgan failure arising from acute kidney injury is reviewed. A second therapeutic pathway, cell processing, does not incorporate stem/progenitor cells in the device, but rather biomimetic materials that remove and modulate the primary cellular components, which promote the worsening organ tissue injury associated with inflammation. The use of an immunomodulating leukocyte selective cytopheretic inhibitory device is also reviewed as an example of this cell processing approach. Both of these unconventional strategies have shown early clinical efficacy in pilot clinical trials and may transform the therapeutic approach to organ failure disorders.

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances, as the isolation of urinary cells is simple (30 ml of urine are sufficient), cost-effective and universal (can be applied to any age, gender and race). Moreover, the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential, and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic, it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study, we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary, the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies.

The supply of human hepatic stem cells (hHpSCs) and other hepatic progenitors has been constrained by the limited availability of liver tissues from surgical resections, the rejected organs from organ donation programs, and the need to use cells immediately. To facilitate accessibility to these precious tissue resources, we have established an effective method for serum-free cryopreservation of the cells, allowing them to be stockpiled and stored for use as an off-the-shelf product for experimental or clinical programs. The method involves use of buffers, some serum-free, designed for cryopreservation and further supplemented with hyaluronans (HA) that preserve adhesion mechanisms facilitating postthaw culturing of the cells and preservation of functions. Multiple cryopreservation buffers were found to yield high viabilities (80-90%) of cells on thawing of the progenitor cells. Serum-free CS10 supplemented with 0.05% hyaluronan proved the most effective, both in terms of viabilities of cells on thawing and in yielding cell attachment and formation of expanding colonies of cells that stably maintain the stem/progenitor cell phenotype. Buffers to which 0.05 or 0.1% HAs were added showed cells postthaw to be phenotypically stable as stem/progenitors, as well as having a high efficiency of attachment and expansion in culture. Success correlated with improved expression of adhesion molecules, particularly CD44, the hyaluronan receptor, E-cadherin, β4 integrin in hHpSCs, and β1 integrins in hepatoblasts. The improved methods in cryopreservation offer more efficient strategies for stem cell banking in both research and potential therapy applications.

In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs.

BACKGROUND Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor, BioLife Solutions, Inc.), the rate of addition of two cryopreservation solutions to cord blood units (CBUs), and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e., slow addition) or as a bolus injection (n = 6; i.e., fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays, total nucleated cells, and CD34+ cell counts were compared. RESULTS Maximum temperature excursions observed were less than 6°C, regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e., final concentration % 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time, formulation consistency, and reduced DMSO in the frozen product (% 5%).

Achievements in tissue engineering using mesenchymal stem cells (MSC) demand a clinically acceptable off-the-shelf" cell therapy product. Efficacy of cryopreservation of human bone marrow-derived MSC in clinically safe animal product-free medium containing 2% 5% and 10% dimethyl sulfoxide (DMSO) was evaluated by measuring cell recovery viability apoptosis proliferation rate expression of a broad panel of MSC markers and osteogenic differentiation. Rate-controlled freezing in CryoStor media was performed in a programmable cell freezer. About 95% of frozen cells were recovered as live cells after freezing in CryoStor solutions with 5% and 10% DMSO followed by storage in liquid nitrogen for 1 month. Cell recovery after 5 months storage was 72% and 80% for 5% and 10% DMSO respectively. Measurements of caspase 3 activity demonstrated that 15.5% and 12.8% of cells after 1 month and 18.3% and 12.9% of cells after 5 months storage in 5% and 10% DMSO respectively were apoptotic. Proliferation of MSC recovered after cryopreservation was measured during 2 weeks post-plating. Proliferation rate was not compromised and was even enhanced. Cryopreservation did not alter expression of MSC markers. Quantitative analysis of alkaline phosphatase (ALP) activity ALP surface expression and Ca deposition in previously cryopreserved MSC and then differentiated for 3 weeks in osteogenic medium demonstrated the same degree of osteogenic differentiation as in unfrozen parallel cultures. Cell viability and functional parameters were analyzed in MSC after short-term storage at 4°C in HypoThermosol-FRS solution also free of animal products. Hypothermic storage for 2 and 4 days resulted in about 100% and 85% cell recovery respectively less than 10% of apoptotic cells and normal proliferation marker expression and osteogenic potential. Overall our results demonstrate that human MSC could be successfully cryopreserved for banking and clinical applications and delivered to the bedside in clinically safe protective reagents.

The availability of human cardiomyocytes derived from embryonic stem cells (ESCs) has generated -considerable excitement, as these cells are an excellent model system for studying myocardial development and may have eventual application in cell-based cardiac repair. Cardiomyocytes derived from the related induced pluripotent stem cells (iPSCs) have similar properties, but also offer the prospects of patient-specific disease modeling and cell therapies. Unfortunately, the methods by which cardiomyocytes have been historically generated from pluripotent stem cells are unreliable and typically result in preparations of low cardiac purity (typically textless1% cardiomyocytes). We detail here the methods for a recently reported directed cardiac differentiation protocol, which involves the serial application of two growth factors known to be involved in early embryonic heart development, activin A, and bone morphogenetic protein-4 (BMP-4). This protocol reliably yields preparations of 30-60% cardiomyocytes, which can then be further enriched to textgreater90% cardiomyocytes using straightforward physical methods.

AIM Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study, we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture, and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition, the cell growth and differentiation process was adaptable to large culture formats. Moreover, the cardiomyocytes survived following cryopreservation, and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium, a step forward towards the application of these cells to human clinical use or drug discovery.

BACKGROUND The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model. METHODOLOGY/PRINCIPAL FINDINGS For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. CONCLUSIONS/SIGNIFICANCE The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza.

Regulatory T cells (Tregs) are a suppressive subset of CD4(+) T lymphocytes implicated in the prevention of acute GVHD (aGVHD) after allo-SCT (ASCT). To determine whether increased frequency of Tregs with a skin-homing (cutaneous lymphocyte Ag, CLA(+)) or a gut-homing (α(4)β(7)(+)) phenotype is associated with reduced risk of skin or gut aGVHD, respectively, we quantified circulating CLA(+) or α(4)β(7)(+) on Tregs at the time of neutrophil engraftment in 43 patients undergoing ASCT. Increased CLA(+) Tregs at engraftment was associated with the prevention of skin aGVHD (2.6 vs 1.7%; P=0.038 (no skin aGVHD vs skin aGVHD)), and increased frequencies of CLA(+) and α(4)β(7)(+) Tregs were negatively correlated with severity of skin aGVHD (odds ratio (OR), 0.67; 95% confidence interval (CI), 0.46-0.98; P=0.041) or gut aGVHD (OR, 0.93; 95% CI, 0.88-0.99; P=0.031), respectively. This initial report suggests that Treg tissue-homing subsets help to regulate organ-specific risk and severity of aGVHD after human ASCT. These results need to be validated in a larger, multicenter cohort.

Human amnion epithelial cells (hAECs) are a heterologous population positive for stem cell markers; they display multilineage differentiation potential, differentiating into cells of the endoderm (liver, lung epithelium), mesoderm (bone, fat), and ectoderm (neural cells). They have a low immunogenic profile and possess potent immunosuppressive properties. Hence, hAECs may be a valuable source of cells for cell therapy. This unit describes an efficient and effective method of hAEC isolation, culture, and cryopreservation that is animal product-free and in accordance with current guidelines on preparation of cells for clinical use. Cells isolated using this method were characterized after 5 passages by analysis of karyotype, cell cycle distribution, and changes in telomere length. The differentiation potential of hAECs isolated using this animal product-free method was demonstrated by differentiation into lineages of the three primary germ layers and expression of lineage-specific markers analyzed by PCR, immunocytochemistry, and histology.