Cell Medicine

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from porcine pancreas. In this study, we evaluated the methods for making a continuous density gradient and loading tissue. One method involved loading digested tissue on top of a continuous density gradient (top loading). The other method involved mixing digested tissue with low-density solution and then making a continuous gradient (mixed loading). There were no significant differences between the 2 purification methods in terms of the islet yield, rate of viability or purity, score, or in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these 2 methods of islet purification.

Stroke is an important cause of morbidity and mortality in children. Clinical presentation is diverse, and multiple risk factors have been described. The aim of this retrospective study is to describe the clinical presentation, risk factors, and the Pediatric National Institute of Health Stroke Scale (PedNIHSS) in a series of pediatric Chilean patients with the diagnosis of arterial ischemic stroke (AIS). Children diagnosed with AIS aged between 29 d and 18 y were enrolled (1989 to 2016). Clinical characteristics and risk factors were described. PedNIHSS severity score was estimated for patients older than 4 mo of age. Sixty-two patients were included, 66% were male, and the mean age of presentation was 3.5 y. Seventy-nine percent presented motor deficit, 45% seizures, and 15% consciousness impairment. Eighty-two percent had a unilateral stroke and 73% had anterior circulation territory affected. The main risk factors were arteriopathy (63%) and infection (43%). The PedNIHSS mean was 7.6, ranging between 0 and 17. In the categories in which it was possible to apply χ² test, only the acute systemic conditions category was statistically significant (P = 0.03), being higher in the group of patients younger than 3 y old. We confirmed male predominance in AIS and the most frequent presenting symptom was motor deficit. We found at least 1 risk factor in all patients with complete information. We confirmed arteriopathy as the most frequent risk factor, and acute systemic conditions were higher in patients younger than 3 y old with statistical significance (P = 0.03). The majority of patients presented mild to moderate severity in the PedNIHSS score.

Across scientific disciplines, 3-D organoid culture systems offer platforms to integrate basic research findings with clinical care. The National Eye Institute mounted a $1.1 million 3-D Retina Organoid Challenge. Organoids developed through the Challenge will be valuable resources for drug screening, disease modeling, and precision and regenerative medicine.

Positioning single cells on a solid surface is a crucial technique for understanding the cellular functions and cell–cell interactions in cell culture assays. We developed a microfluidic chip for depositing single cells in microwells using a simple micropipette operation. Cells were delivered to microwells by the meniscus motion of liquid interface. The residue deposits of cells were redistributed with air injection, and the isolated single cells were stored in microwells. Different microwell sizes and depths were studied to evaluate the trapping possibility of cells. Medium replacement and cell viability staining with the isolated single cells were achieved in microwells. The chip will serve as a tool for single-cell patterning in an easy-to-use manner.

The tumor microenvironment can be realistically viewed as an active battle ground between the host immune system and the growing tumor cells. This reactive space surrounding the tumor possesses several possibilities and facilitates the progression of a tumor from a neoplastic stage to that of metastasis. The contemporary approach of understanding the cancer biology from a “within the cell” perspective has been largely challenged with complex and intricate “outside the cell” events. Thus understanding the biology of the tumor microenvironment has been of scientific and clinical interest. Small non-coding microRNAs with a pleotropic and wide range of cellular gene targets can be reasonably hypothesized to regulate the events of carcinogenesis and progression. MicroRNAs have been investigated in different cancer models, and evidence of their involvement in the regulation of the tumor microenvironment has been of much interest. In particular, a major interest has been exploring the role of the tumor microenvironment in regulating the interaction of cancer cells with surrounding stromal components and the effect of such interactions on the cancer cells. Fine-tuned regulation by these microRNAs extends our contemporary understanding of these small biomolecules in epigenetic regulations. This review focuses on microRNAs that are dysregulated in ovarian carcinomas, their effect on the components of the tumor microenvironment, and the correlation of their heterogeneous expression profiles with disease severity and prognosis in patients. In addition, this paper also discusses the differential expression of exosomal microRNAs that are known to link the cancer cell with its microenvironment, facilitating the development of an improved prognostic/diagnostic marker and effective therapeutic regime.

Several techniques exist to manage bone defects in patients: bone grafts (autograft, allograft, xenograft), use of synthetic bone substitutes, or use of the products of bone regenerative medicine. Studies generally focus on their efficacy, but few focus on their acceptance. Our objectives were to assess their theoretical acceptance among the French general population, and to identify issues justifying refusals, by mean of an open e-questionnaire. The questionnaire was submitted to a general French population, and explained these techniques in an understandable way. Participants were asked to say whether they would accept or refuse these techniques, specifying why in case of refusal (fear of the technique, ethical reasons, religious reasons). In total, 562 persons participated. Autograft and use of the products of bone regenerative medicine were the most accepted techniques (93.4% and 94.1%, respectively). Xenograft was the least accepted technique (58.2%). Most refusals were due to fear such as failure, pain, infection (autograft 8%, allograft 14.9%, xenograft 25.3%, synthetic bone substitutes 14.6%, and products of bone regenerative medicine 6.8%). Ethical reasons were mostly mentioned for allograft (6.4%) and xenograft (18.3%). Religious reasons were scarcely mentioned, only for xenograft (1.2%). Thus, acceptance of techniques does not seem to be greatly linked to sociodemographic characteristics in France. However, other countries with their own cultural, religious, and population patterns may show different levels of acceptance. This study shows that bone regenerative medicine is a promising research direction, reaching biological and also humanist quality standards, expected to improve the health of patients. Information is still the cornerstone to defuse issues about fear.

Purification of pancreatic islets is an important step in islet isolation for islet transplantation. In this study, to investigate how a solution composed mainly of Na-lactobionate and histidine (HL) influences the purification of islets, iodixanol was added to a purified solution for porcine islet isolation. A solution (IU) made by adding iodixanol to University of Wisconsin solution and a solution (IHL) made by adding iodixanol to HL solution were used to evaluate the islet isolation performance. We noted no significant differences between the two purification methods with regard to the islet yield, survival rate or purity, score, or stimulation index. These results show that IHL solution is as useful as IU solution for islet purification.

Freezing is recognized as the most effective method of maintaining a stable supply of various cell types for long-term storage. However, cells might be damaged by environmental changes during the freezing process. There are various factors that influence the function of cells cultured after cryopreservation and thawing. These factors include cryopreservation solutions, biomaterials, freezing methods, and the freezing and preservation temperatures. There is also a risk of infection with mycoplasma in liquid nitrogen phase. Therefore, it is necessary to consider more useful and safe methods for freezing and storing various cells. In this study, we investigated the effects of temperature during long-term storage (8 years at −80 °C and in liquid nitrogen phase) on the quality of various cells (human hepatocellular carcinoma cells, bovine carotid artery normal endothelial cells, mouse fibroblast cells 3T3, and mouse embryo fibroblast cells STO). We examined the cell viability of cryopreserved human hepatocellular carcinoma cells at −80 °C using culture medium containing 10% DMSO, Cell Banker 1, and Cell Banker 2 as cryopreservation solutions. Among these solutions, Cell Banker 1 showed the highest efficiency. The viability of human hepatocellular carcinoma and bovine carotid artery normal endothelial cells in the Cell Banker 1 stored at −80 °C was over 90%, which was the same as that in liquid nitrogen phase. The cells stored at −80 °C had a morphology similar to that of the cells stored at liquid nitrogen phase. The proliferation of cells stored at −80 °C and in liquid nitrogen phase was not significantly different. Furthermore, none of the cells were infected with mycoplasma. There was no marked difference in the albumin secretion between the human hepatocellular carcinoma cells stored at −80 °C and those in liquid nitrogen phase. The short tandem repeats of the human hepatocellular carcinoma cells stored at −80 °C were identical to those stored in liquid nitrogen phase. In this report, various cells stored long-term at −80 °C were able to be used effectively after long-term storage. These findings can be applied to drug discovery, cell medicine, and cell therapy.

Islet transplantation has been shown to restore normoglycemia clinically. One of the current limitations to the widespread clinical use of islet transplantation is culturing and preserving more than 1 million islet equivalents in preparation for transplant. One possible solution is to bank frozen islets and use them when needed. Although promising, the standard islet freezing protocol introduces stress and cell death, resulting in high variability of islet quality post thawing. This study aimed to develop an improved cryopreservation protocol using alginate-encapsulated islets to improve islet survival and function for future transplants. Our data showed that encapsulation improved islet survival and function after thawing the frozen islets. Frozen encapsulated islets have an islet yield recovery of 84% when compared to non-encapsulated islets at 72% after thawing. Post-thaw viability was 78% for non-encapsulated islets compared to 88% for encapsulated islets. The stimulation index values after a static glucose test following thawing were 1.9 ± 0.5, 2.9 ± 0.1, and 3.3 ± 0.3 for the non-encapsulated, 1.75% alginate, and 2.5% alginate groups, respectively. In a transplant study, the mice that received 1.75% alginate-encapsulated cryopreserved islets achieved normoglycemia on average 5 days after transplant. In comparison, control mice that received fresh islets took 4 days, while those receiving unencapsulated cryopreserved islets took 18 days. In conclusion, encapsulating islets in 1.75% alginate prior to freezing was shown to improve islet survival, function post thawing, and graft response significantly when compared to islets frozen without encapsulation.

The liver is currently the site of choice for clinical islet transplantation, even though many alternative implantation sites have lately been proposed as more ideal for graft survival. The suggested sites, for example intramuscular space, omentum, bone marrow, and spleen, are sometimes difficult to compare due to differences in animal model, islet isolation procedure, and islet quality. In addition, the variation in transplanted islet mass is vast. The aim of this commentary is to review alternative implantation sites tested experimentally as well as in clinical islet transplantation. Although many sites have been investigated, none have convincingly proved better suited for clinical islet transplantation than intraportal injection to the liver, regardless of whether it is autologous or allogeneic transplantation. However, in order to fully evaluate upcoming bioengineering techniques, such as scaffolds containing insulin-producing cells derived from stem cells, the need of an alternative site has arisen to enable cellular monitoring, which currently cannot be achieved within the liver.

Ischemic–reperfusion (I/R) injury to cerebral white matter during the perinatal period leads to long-term cognitive and motor disabilities in children. Immature white matter oligodendrocytes are especially vulnerable to metabolic insults such as those caused by hypoxic, ischemic, and reperfusion injury. Consequences include an impaired capacity of oligodendrocytes to generate and maintain mature lipid-rich myelin needed for efficient neuronal conductivity. Further research is needed to increase an understanding of the early, possibly reversible myelin-associated pathologies that accompany I/R white matter injury. This experiment characterized I/R time-dependent alterations in cerebral white matter lipid profiles in an established fetal sheep model. Fetal sheep (127 days gestation) were subjected to 30 min of bilateral carotid artery occlusion followed by 4 h (n = 5), 24 h (n = 7), 48 h (n = 3), or 72 h (n = 5) of reperfusion, or sham treatment (n = 5). Supraventricular cerebral white matter lipids were analyzed using the positive ionization mode matrix-assisted laser desorption/ionization mass spectrometry. Striking I/R-associated shifts in phospholipid (PL) and sphingolipid expression with a prominent upregulation of cardiolipin, phosphatidylcholine, phosphatidylinositol monomannoside, sphingomyelin, sulfatide, and ambiguous or unidentified lipids were observed to occur mainly at I/R-48 and normalized or suppressed responses at I/R-72. In fetal sheep, cerebral I/R caused major shifts in white matter myelin lipid composition favoring the upregulated expression of diverse PLs and sphingolipids which are needed to support neuronal membrane, synaptic, metabolic, and cell signaling functions.

Safety concerns over cell-derived pharmaceutical products being manufactured in supplements of fetal bovine serum (FBS) have ignited pleas to replace FBS. Herein, four newly marketed alternatives to FBS were compared: a xeno-free product called Cell-Ess®, a human platelet lysate marketed as GroPro®, and two mixtures of adult bovine serum varying in their proportions of neonatal growth factors, called Liporo® and FetalGro®. An endothelial cell line (C2BBe1) and a neuronal cell line (SHSY5Y) near confluency in media with 10% FBS were selectively scraped and taken through a 25-day step-wise algorithm to replace FBS, and another human endothelial cell line (HRA-19) was studied to replicate C2BBe1. Cells were stained, counted, and compared for viability, migration, and spheroids. The C2BBe1 and HRA-19 cell lines failed to proliferate in 10% Cell-Ess® but grew in 10% GroPro® or 10% FetalGro® reasonably well compared to reference 10% FBS. With SH-SY5Y, only FetalGro® approached FBS's efficacy. These were all inferior to 11 different branded lots of FBS (positive controls), but five days into switching just amongst the FBS brands, 4 of 11 supported less proliferation than reference FBS in endothelial HRA-19 (p < 0.004). Moreover, neurospheres were enriched in two branded lots of FBS and FetalGro® (each p < 0.004), neurospheres being an unwanted phenotype for any neuronal cell application. Because platelet-derived GroPro® stood out amongst the non-FBS growth supplements to allow proliferation without inducing spheroids, it seems the best (mindful that the cells still grew slower in it compared to FBS). While no perfect replacement was found amongst the alternatives to FBS, the algorithm for switching should be useful in future testing of new alternatives to FBS as the need arises to switch from FBS and expand pharmaceutical products with safety for human use.

In the United States, Alzheimer's disease (AD) is the most common cause of dementia, accompanied by substantial economic and emotional costs. During 2015, more than 15 million family members who provided care to AD patients had an estimated total cost of 221 billion dollars. Recent studies have shown that elevated total plasma levels of homocysteine (tHcy), a condition known as hyperhomocysteinemia (HHcy), is a risk factor for AD. HHcy is associated with cognitive decline, brain atrophy, and dementia; enhances the vulnerability of neurons to oxidative injury; and damages the blood–brain barrier. Many therapeutic supplements containing vitamin B12 and folate have been studied to help decrease tHcy to a certain degree. However, a therapeutic cocktail approach with 5-methyltetrahydrofolate, methyl B12, betaine, and N-acetylcysteine (NAC) have not been studied. This novel approach may help target multiple pathways simultaneously to decrease tHcy and its toxicity substantially.

The purification step is one of the most important and difficult procedures in islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from the porcine pancreas. In this study, we evaluated the impact of the timing of tissue loading on porcine islet purification using large plastic bottles. One method involved loading digested tissue after creating a continuous density gradient (tissue after gradient [TAG]). The other method involved loading digested tissue before creating a continuous density gradient (tissue before gradient [TBG]). There were no significant differences between TAG and TBG in terms of the islet yield, rates of viability and purity, score, and in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of post-transplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

The low efficiency of in vitro differentiation of human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (iPSCs) into insulin-producing cells is a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). Our previous investigation into the key factors for the differentiation of PSCs into insulin-producing cells suggested that the expression of GATA binding protein 6 (GATA6) and Gremlin 1 (GREM1) and inhibition of early growth response protein 1 (Egr1) may be important factors. In this study, we investigated the role of Egr1 in pancreas development. The transfection of small interfering RNA (siRNA) of Egr1 in the early phase induced the differentiation of iPSCs derived from fibroblasts (FiPSCs) into pancreatic endoderm and insulin-producing cells. In contrast, the downregulation of Egr1 in the late phase suppressed the differentiation of FiPSCs into pancreatic endoderm and insulin-producing cells. In addition, the overexpression of Egr1 suppressed the differentiation of iPSCs derived from pancreatic cells into pancreatic endoderm and insulin-producing cells. These data suggest that the downregulation of Egr1 in the early phase can efficiently induce the differentiation of iPSCs into insulin-producing cells.

Bronchopulmonary dysplasia (BPD), a disease affecting extremely premature infants, results from the disruption of normal pulmonary vascular and alveolar growth. Currently, there is no specific effective treatment. We report a case of a 10-mo-old female infant with BPD, who was admitted because of adenovirus pneumonia and acute respiratory distress syndrome (ARDS) with prolonged venovenous and arteriovenous extracorporeal membrane oxygenation (ECMO) support (total 125 d). The respiratory condition dramatically improved, and ECMO was removed 25 d after intratracheal delivery of maternal bone marrow-derived mesenchymal stem cells (BM-MSCs). Short tandem repeat examinations revealed that there was no maternal cells in the bronchial wash fluid. To our knowledge, this is the first human report of BM-MSC therapy reversal of the course of BPD superimposed with ARDS. We also suggest that BM-MSC therapy may not only be effective in the newborn stage but also works in infants and children with BPD.

During development, stem cells generate all of the differentiated cells that populate our tissues and organs. Stem cells are also responsible for tissue turnover and repair in adults, and as such, they hold tremendous promise for regenerative therapy. Aging, however, impairs the function of stem cells and is thus a significant roadblock to using stem cells for therapy. Paradoxically, the patients who would benefit the most from regenerative therapies are usually advanced in age. The use of stem cells from young donors or the rejuvenation of aged patient-derived stem cells may represent part of a solution. Nonetheless, the transplantation success of young or rejuvenated stem cells in aged patients is still problematic, since stem cell function is greatly influenced by extrinsic factors that become unsupportive with age. This article briefly reviews how aging impairs stem cell function, and how this has an impact on the use of stem cells for therapy.