Immunoadsorption therapy for dilated cardiomyopathy using tryptophan column—A prospective,multicenter, randomized,within‐patient and parallel‐group comparative study to evaluate efficacy and safety

Over the past few decades, several cardiac autoantibodies have been reported in sera from patients with dilated cardiomyopathy (DCM). Immunoadsorption (IA) therapy is one of the therapeutic tools to remove such autoantibodies. The objective of this study was to investigate functional effects of IA therapy using a tryptophan column in severe DCM patients. Of 49 patients enrolled, 44 were randomized from 10 sites in Japan. IA therapy was conducted in 40 patients with DCM (refractory to standard therapy for heart failure, New York Heart Association [NYHA] class III/IV, left ventricular ejection fraction [LVEF] <30%). Mean echocardiographic LVEF was significantly improved (23.8 ± 1.3% to 25.9 ± 1.3%, P = 0.0015). However, mean radionuclide LVEF over 3 months of IA therapy was not significantly improved (20.8 ± 1.1% to 21.9 ± 1%, P = 0.0605). The cardiothoracic ratio was also significantly decreased (P = 0.0010). NYHA functional class (P < 0.0001), subjective symptoms assessed by a quality of life questionnaire (P = 0.0022), maximum oxygen consumption (P = 0.0074), and 6‐minute walk distance (P = 0.0050) were improved after IA therapy. Subgroup analysis revealed improvement of echocardiographic LVEF in patients with higher baseline autoantibody scores but not in those with lower scores. IA therapy improved subjective symptoms and exercise capacity in patients with refractory heart failure resulting from DCM. Favorable effect on cardiac function was noted in patients with higher autoantibody scores. J. Clin. Apheresis 31:535–544, 2016. © 2015 Wiley Periodicals, Inc.     

Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature‐responsive closed culture device

Temperature‐responsive culture surfaces make it possible to harvest transplantable carrier‐free cell sheets. Here, we applied temperature‐responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony‐forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature‐responsive closed culture devices. The results were similar to those obtained using temperature‐responsive culture inserts. These results indicate that the novel temperature‐responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets. Copyright © 2013 John Wiley & Sons, Ltd.     

Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment

Multipotent haematopoietic stem and progenitor cells (HSPCs) are the source for all blood cell types. The bone marrow stem cell niche in which the HSPCs are maintained is known to be vital for their maintenance. Unfortunately, to date, no in vitro model exists that accurately mimics the aspects of the bone marrow niche and simultaneously allows the long‐term culture of HSPCs. In this study, a novel three‐dimensional coculture model is presented, based on a hydroxyapatite coated zirconium oxide scaffold, comprising of human mesenchymal stromal cells (MSCs) and cord blood derived HSPCs, enabling successful HSPC culture for a time span of 28 days within the microfluidic multiorgan chip. The HSPCs were found to stay in their primitive state (CD34⁺CD38^?) and capable of granulocyte, erythrocyte, macrophage, megakaryocyte colony formation. Furthermore, a microenvironment was formed bearing molecular and structural similarity to the in vivo bone marrow niche containing extracellular matrix and signalling molecules known to play an important role in HSPC homeostasis. Here, a novel human in vitro bone marrow model is presented for the first time, capable of long‐term culture of primitive HSPCs in a microfluidic environment.     

Cell encapsulation within PVA‐based hydrogels via freeze‐thawing: a one‐step scaffold formation and cell storage technique

Cryogelation is a physical hydrogel formation method for certain polymers, notably polyvinyl alcohol (PVA). The hypothesis of this study is that a PVA‐based solution with the necessary intracellular cryoprotectant and nutrient supply can be used, first for storage of vascular smooth muscle cells, and subsequently to form a suitable tissue‐engineering scaffold during the thawing process. Bovine arterial smooth muscle cells were encapsulated within PVA–gelatin hydrogels over a wide range of serum, DMSO and cell culture medium concentrations. Several parameters expected to affect gelation and cell viability (PVA viscosity, DMSO concentration, serum presence) were assessed with experimental designs and the optimal conditions for cell survival were determined. Cell viability can be improved by increasing concentration of DMSO and serum without compromising the gelation process. An additional crosslinking step using a coagulation bath was beneficial for hydrogel stability but caused peripheral accumulation of cells. In conclusion, a freeze–thaw process can be utilized to prepare and store cell‐laden hydrogels with adjustable mechanical properties. Copyright © 2009 John Wiley & Sons, Ltd.     

Regeneration of dental pulp/dentine complex with a three‐dimensional and scaffold‐free stem‐cell sheet‐derived pellet

Dental pulp/dentine complex regeneration is indispensable to the construction of biotissue‐engineered tooth roots and represents a promising approach to therapy for irreversible pulpitis. We used a tissue‐engineering method based on odontogenic stem cells to design a three‐dimensional (3D) and scaffold‐free stem‐cell sheet‐derived pellet (CSDP) with the necessary physical and biological properties. Stem cells were isolated and identified and stem cells from root apical papilla (SCAPs)‐based CSDPs were then fabricated and examined. Compact cell aggregates containing a high proportion of extracellular matrix (ECM) components were observed, and the CSDP culture time was prolonged. The expression of alkaline phosphatase (ALP), dentine sialoprotein (DSPP), bone sialoprotein (BSP) and runt‐related gene 2 (RUNX2) mRNA was higher in CSDPs than in cell sheets (CSs), indicating that CSDPs have greater odonto/osteogenic potential. To further investigate this hypothesis, CSDPs and CSs were inserted into human treated dentine matrix fragments (hTDMFs) and transplanted into the subcutaneous space in the backs of immunodeficient mice, where they were cultured in vivo for 6 weeks. The root space with CSDPs was filled entirely with a dental pulp‐like tissue with well‐established vascularity, and a continuous layer of dentine‐like tissue was deposited onto the existing dentine. A layer of odontoblast‐like cells was found to express DSPP, ALP and BSP, and human mitochondria lined the surface of the newly formed dentine‐like tissue. These results clearly indicate that SCAP‐CSDPs with a mount of endogenous ECM have a strong capacity to form a heterotopic dental pulp/dentine complex in empty root canals; this method can be used in the fabrication of bioengineered dental roots and also provides an alternative treatment approach for pulp disease. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of a human fetal spinal cord stem cell line,NSI‐566RSC,and its induction to functional motoneurons

Specific neuronal subtypes, especially motoneurons (MNs), derived from human stem cells provide a significant therapeutic potential for spinal cord diseases, such as amyotrophic lateral sclerosis (ALS) and spinal cord injury. So far, in vitro, MNs have only been successfully induced from embryonic stem cells (hESC) and human fetal cortical progenitors. Although neural progenitors from spinal cord would be a likely source for generating MNs, there has been no study reporting successful in vitro differentiation of MNs from spinal cord progenitors. This study first characterized a polyclonal spinal cord stem cell line isolated from an 8 week‐old fetus. Then a paradigm was introduced to successfully induce MNs from this cell line, which was demonstrated by immunostaining using the MN markers HB9, Islet1 and choline acetyl transferase (ChAT). The combination of HB9 and ChAT immunostainings indicated that ～20% of the cells were MNs after this induction protocol. The presence of other cell types in the differentiated culture was also analysed. Finally, the electrophysiological properties of these differentiated MNs were characterized to confirm their functional integrity. The majority of these MNs fired repetitive action potentials (APs), which is an indicator of functional maturation. The recordings of spontaneous excitatory postsynaptic currents (EPSCs) confirmed the formation of synapses onto these MNs. This study reports the first successful differentiation of MNs from human spinal cord stem cells in vitro, providing a novel approach for obtaining functional MNs when designing the therapeutic strategy for spinal cord diseases or injuries. Copyright © 2009 John Wiley & Sons, Ltd.     

Hematopoietic progenitor cell mobilization using low‐dose cyclophosphamide and granulocyte colony‐stimulating factor for multiple myeloma

High‐dose chemotherapy (HDT) supported by autologous stem cell transplantation (ASCT) has long been one of the standards of care for younger patients with multiple myeloma (MM). Cyclophosphamide (CY) plus granulocyte colony‐stimulating factor (G‐CSF) has been the conventional preparation for hematopoietic progenitor cell (HPC) mobilization, although the optimal dosage of CY in this setting has not yet been clearly defined. This study investigated the efficacy and safety of low‐dose (LD‐)CY (1.5 g/m²) plus G‐CSF for conditioning for HPC apheresis harvest (HPC‐A) in 18 MM patients, and compared it with a regimen consisting of intermediate‐dose (ID)‐CY (4 g/m²) plus G‐CSF for 13 MM patients. Eleven patients in the former and six in the latter were treated with bortezomib (BTZ) during the induction therapy. Both regimens were comparably effective in terms of CD34⁺ cell yields, while adverse events, such as leukopenia, thrombocytopenia, and febrile neutropenia, occurred significantly less frequently in the LD‐CY cohort. All patients in LD‐CY cohort started and completed their apheresis on day 7 or 8, whereas for the ID‐CY cohort the day of first apheresis varied widely from day 8 to 15. These findings indicate that the LD‐CY regimen is as effective as ID‐CY for HPC mobilization, while the former is clearly more practicable and convenient than the ID‐CY regimen for patients with MM. J. Clin. Apheresis 28:368–373, 2013. © 2013 Wiley Periodicals, Inc.     

In vivo quantitative assessment of cell viability of gadolinium or iron‐labeled cells using MRI and bioluminescence imaging

In cell therapy, noninvasive monitoring of in vivo cell fate is challenging. In this study we investigated possible differences in R₁, R₂ or R₂* relaxation rate as a measure of overall cell viability for mesenchymal stem cells labeled with Gd‐liposomes (Gd‐MSCs) or iron oxide nanoparticles (SPIO‐MSCs). Cells were also transduced with a luciferase vector, facilitating a correlation between MRI findings and cell viability using bioluminescence imaging (BLI). Viable Gd‐MSCs were clearly distinguishable from nonviable Gd‐MSCs under both in vitro and in vivo conditions, clearly differing quantitatively (ΔR₁ and ΔR₂) as well as by visual appearance (hypo‐ or hyperintense contrast). Immediately post‐injection,viable Gd‐MSCs caused a substantially larger ΔR₂ and lower ΔR₁ effect compared with nonviable MSCs. With time, the ΔR₁ and ΔR₂ relaxation rate showed a good negative correlation with increasing cell number following proliferation. Upon injection, no substantial quantitative or visual differences between viable and nonviable SPIO‐MSCs were detected. Moreover, nonviable SPIO‐MSCs caused a persisting signal void in vivo, compromising the specificity of this contrast agent. In vivo persistence of SPIO particles was confirmed by histological staining. A large difference was found between SPIO‐ and Gd‐labeled cells in the accuracy of MR relaxometry in assessing the cell viability status. Gd‐liposomes provide a more accurate and specific assessment of cell viability than SPIO particles. Viable Gd cells can be differentiated from nonviable Gd cells even by visual interpretation. These findings clearly indicate Gd to be the favourable contrast agent in qualitative and quantitative evaluation of labeled cell fate in future cell therapy experiments. Copyright © 2012 John Wiley & Sons, Ltd.     

Allyl‐isatin suppresses cell viability,induces cell cycle arrest,and promotes cell apoptosis in hepatocellular carcinoma HepG2 cells

The anticancer effect of the newly synthesized isatin derivative, N‐allyl‐isatin (Allyl‐I), was evaluated in vitro with human hepatocellular carcinoma HepG2 cells. Cell viability was detected by cell counting kit‐8 (CCK8) assay. Acridine orange (AO)/ethidium bromide (EB) double staining was used to observe the cell morphology. Flow cytometry was used to assess the effects of Allyl‐I on the cell cycle, apoptosis rate, and mitochondrial membrane potential (MMP). Western blot analysis was performed to detect the influence of Ally1‐I on the expression of cytochrome c (cyt c), Bax, Bcl‐2, and cleaved caspase‐3. Allyl‐I significantly inhibited HepG2 cell viability in a time‐ and dose‐dependent manner. Allyl‐I can induce cell cycle arrest in HepG2 cells at the G2/M phase. Apoptotic nuclear morphological changes were observed after AO/EB double staining. Fluorescein isothiocyanate‐conjugated Annexin V (Annexin V‐FITC) and propidium iodide (PI) double staining showed that the apoptotic rates significantly increased in the presence of Allyl‐I. Rhodamine 123 staining indicated that Allyl‐I can decrease the MMP. Allyl‐I also altered the expression of mitochondrial apoptosis‐related proteins. Protein levels of cyt c and cleaved caspase‐3 were upregulated following Allyl‐I treatment. By contrast, the Bcl‐2/Bax ratio decreased. Results suggest that Allyl‐I suppresses cell viability, induces cell cycle arrest, and promotes cell apoptosis in HepG2 cells. Furthermore, the induction of apoptosis might be correlated with the mitochondrial pathway.     

Efficacy and safety of lornoxicam compared with placebo and diclofenac in acute sciatica/lumbo‐sciatica: an analysis from a randomised,double‐blind,multicentre, parallel‐group study

Aim: The efficacy and safety of oral lornoxicam (LNX) as early treatment of acute sciatica/lumbo‐sciatica was compared with placebo and diclofenac in a 5‐day double‐blind, randomised study. Methods: Male or female patients (n = 171) aged 18–70 years with acute sciatica or lumbo‐sciatica [acute sciatica defined as typical radiation of pain along the sciatic nerve (including radiating pain below the knee) and worsening of pain as defined by Lasegue’s leg‐raising test (< 60°) within 72 h and previous attack ceased > 3 months previously; lumbo‐sciatica defined as symptoms of sciatica with concurrent lumbar pain and a predefined minimum pain score]. The dosage of study treatment was 8–24 mg/day LNX, 100–150 mg/day diclofenac or placebo. The primary end‐point was the difference in pain intensity difference from baseline to 6 h (PID_{0–6 h}) after the first dose of study treatment. Secondary end‐points were pain relief, the cumulative sums of pain intensity difference and total pain relief on day 1 and on days 2–4. Results: In total, 164 patients completed the study. Significant differences in PID between LNX and placebo were seen in the time interval 3–8 h after the first dose including PID_{0–6 h} (p = 0.015). Secondary end‐points favoured LNX vs. placebo, but in general were not significantly different. LNX and diclofenac had similar analgesic effect. Incidence and severity of adverse events were comparable for the three treatments; overall tolerability was rated as very good/good by 93% of the patients. Conclusion: These data indicate that the analgesic efficacy of LNX is superior to placebo and similar to diclofenac in acute sciatica/lumbo‐sciatica.     

Splenic rupture in a plasma cell leukemia,mobilized with G‐CSF for autologous stem cell transplant

Splenic rupture (SR) is a rare adverse event observed in patients treated with G‐CSF as a peripheral hematopoietic stem cell (PHSC) mobilizing agent, mostly in myeloma multiple and amiloidosis; to date, to our knowledge, it has not been previously described in plasma‐cell leukemia (PCL). We report a case of a woman with PCL, who presented a SR after PHSC mobilization with Cyclophosphamide+G‐CSF. The spleen removed showed hematopoietic foci and amiloid material. In the course of a second mobilization, 2 months after, the patient died from sepsis. We considered it important to report this case, in order to keep in mind the possibility of SR in patients with malignant gammopathy. J. Clin. Apheresis 25:223–225, 2010. © 2010 Wiley‐Liss, Inc.     

In vivo construction of liver tissue by implantation of a hepatic non‐parenchymal/adipose‐derived stem cell sheet

Subcutaneous hepatocyte sheet implantation is an attractive therapeutic option for various liver diseases. However, this technique is limited by the availability of hepatocytes. Thus, the use of hepatic non‐parenchymal cells (NPCs) containing small hepatocytes, which have the ability to proliferate more rapidly than mature hepatocytes, for transplantation has been suggested. The aim of our study was to construct liver tissue subcutaneously in rats by implanting NPC sheets co‐cultivated with adipose‐derived stem cells (ADSCs), which produce certain angiogenic factors. We crafted NPC‐ADSC sheets on temperature‐responsive culture dishes. NPCs formed functioning bile canaliculi and stored glycogen. In addition, their ability to produce albumin was not inferior to that of hepatocytes. Albumin production increased over time when co‐cultivated with ADSCs. We then implanted the co‐cultivated cell sheets subcutaneously. The co‐cultivated sheets retained glycogen, formed bile canaliculi, showed signs of vascularization and survived subcutaneously without pre‐vascularization. These results suggest that NPCs can be a viable option in cell therapy for liver diseases. This technique using co‐cultivated cell sheets may be useful in the field of regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.     

Online measurement of oxygen enables continuous noninvasive evaluation of human‐induced pluripotent stem cell (hiPSC) culture in a perfused 3D hollow‐fiber bioreactor

For clinical and/or pharmaceutical use of human‐induced pluripotent stem cells (hiPSCs), large cell quantities of high quality are demanded. Therefore, we combined the expansion of hiPSCs in closed, perfusion‐based 3D bioreactors with noninvasive online monitoring of oxygen as culture control mechanism. Bioreactors with a cell compartment volume of 3 or 17 ml were inoculated with either 10 × 10⁶ or 50 × 10⁶ cells, and cells were expanded over 15 days with online oxygen and offline glucose and lactate measurements being performed. The CellTiter‐Blue® Assay was performed at the end of the bioreactor experiments for indirect cell quantification. Model simulations enabled an estimation of cell numbers based on kinetic equations and experimental data during the 15‐day bioreactor cultures. Calculated oxygen uptake rates (OUR), glucose consumption rates (GCR), and lactate production rates (LPR) revealed a highly significant correlation (p < 0.0001). Oxygen consumption, which was measured at the beginning and the end of the experiment, showed a strong culture growth in line with the OUR and GCR data. Furthermore, the yield coefficient of lactate from glucose and the OUR to GCR ratio revealed a shift from nonoxidative to oxidative metabolism. The presented results indicate that oxygen is equally as applicable as parameter for hiPSC expansion as glucose while providing an accurate real‐time impression of hiPSC culture development. Additionally, oxygen measurements inform about the metabolic state of the cells. Thus, the use of oxygen online monitoring for culture control facilitates the translation of hiPSC use to the clinical setting.     

[18F]‐FBEM,a tracer targeting cell‐surface protein thiols for cell trafficking imaging

We used [¹⁸F]‐4‐fluorobenzamido‐N‐ethylamino‐maleimide ([¹⁸F]‐FBEM) to radiolabel cells ex vivo for in vivo positron emission tomography (PET) in order to assess cell trafficking in mice. In contrast to commonly used imaging agents, [¹⁸F]‐FBEM forms a covalent bond with thiol groups present on the cells surface. The stability of the probe in aqueous medium was tested at different pH values and cross‐experiment showed that thiol‐labeling efficiency was retained (at least) up to pH 9. The labeling procedure did not affect significantly the cell viability. To illustrate the procedure, PET images of living mice injected intravenously with labeled T lymphocytes were obtained. They showed the expected cell homing in the spleen that was absent in mice injected with free label. Copyright © 2013 John Wiley & Sons, Ltd.     

Successful reprogramming of differentiated cells by somatic cell nuclear transfer,using in vitro‐matured oocytes with a modified activation method

Therapeutic cloning has tremendous potential for cell therapy and tissue repair in some diseases. However, the efficiency of development of cloned human embryos by somatic cell nuclear transfer is still low. In the present study, the activation of cloned human embryos was investigated while using in vitro‐matured oocytes. Pseudo‐pronuclear formation and the subsequent development was compared with different activation parameters, including different durations of ionomycin and 6‐dimethylaminopurine treatment. The results showed that somatic cells were successfully reprogrammed by modification of activation treatments while using in vitro‐matured oocytes. The activation efficiency of cloned human embryos was significantly increased at durations of ionomycin at both 5 and 7 min, despite different durations of 6‐DMAP treatment. The results of blastocyst development showed that 20% of activated embryos developed to the blastocyst stage when the embryos were activated with 5 µm ionomycin for 5 min and 2 mm 6‐DMAP for 5 h, which was significantly higher than those activated with other parameters. Moreover, we found that an increasing duration of 6‐DMAP induced the formation of a single, large, pseudo‐pronucleus in cloned human embryos and impaired subsequent development competence. In conclusion, successful reprogramming of human somatic cells was achieved using in vitro‐matured oocytes by somatic cell nuclear transfer and improved with a modified activation method. Copyright © 2012 John Wiley & Sons, Ltd.