Translating stem cell research into development of cellular drugs-a perspective from manufacture of stem cell products and CMC considerations.

The development of human pluripotent stem cell (PSC)-derived medicinal products has been gathering steam in recent years, but the translation of research protocols into GMP production remains a daunting task. The challenges not only reside with the nature of cellular therapeutics but are also rooted in the general inexperience in industry-scale production of stem cell products. Manufacturers of PSC-derived products should be aware of the technical nuances and take a holistic approach toward early planning and engagement with their academic partners. While not all issues will be readily resolved soon, the collective knowledge and consensus by the manufacturers and key stakeholders will help to guide rapid progression of the field.

Aging, rather than Parkinson's disease, affects the responsiveness of PBMCs to the immunosuppression of bone marrow mesenchymal stem cells.

Whether aging or Parkinson's disease (PD) affects the responses of peripheral blood mononuclear cells (PBMCs) to immunosuppression by bone marrow­derived mesenchymal stem cell (BM­MSCs) and which cytokines are more effective in inducing BM­MSCs to be immunosuppressive remains to be elucidated. PBMCs were isolated from healthy young (age 26­35), healthy middle­aged (age 56­60) and middle­aged PD­affected individuals. All the recruits were male. The mitogen­stimulated PBMCs and proinflammatory cytokine­pretreated BM­MSCs were co­cultured. The PBMC proliferation was measured using Cell Counting Kit­8, while the cytokine secretion was assayed by cytometric bead array technology. The immunosuppressive ability of BM­MSCs was confirmed in young healthy, middle­aged healthy and middle­aged PD­affected individuals. Among the three groups, the PBMC proliferation and cytokine secretion of the young healthy group were suppressed more significantly compared with those of the middle­aged healthy and middle­aged PD­affected group. No significant differences were identified in the PBMC proliferation and cytokine secretion between the patients with PD and the middle­aged healthy subjects. Interferon (IFN)­Î³ synergized with tumor necrosis factor (TNF)­α, interleukin (IL)­1α or IL­1ß was more effective than either one alone, and the combinations of IFN­Î³ + IL­1α and IFN­Î³ + IL­1ß were more effective than IFN­Î³ + TNF­α in inducing BM­MSCs to inhibit PBMC proliferation. The results of the present study suggested that aging, rather than PD, affects the response of PBMCs toward the suppression of BM­MSC, at least in middle­aged males. Patients with PD aged 56­60 remain eligible for anti­inflammatory BM­MSC­based therapy. Treatment of BM­MSCs with IFN­Î³ + IL­1α or IFN­Î³ + IL­1ß prior to transplantation may result in improved immunosuppressive effects.

Krüppel-like factor 4 (KLF4) induces mitochondrial fusion and increases spare respiratory capacity of human glioblastoma cells.

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor critical for the regulation of many cellular functions in both normal and neoplastic cells. Here, using human glioblastoma cells, we investigated KLF4's effects on cancer cell metabolism. We found that forced KLF4 expression promotes mitochondrial fusion and induces dramatic changes in mitochondrial morphology. To determine the impact of these changes on the cellular functions following, we analyzed how KLF4 alters glioblastoma cell metabolism, including glucose uptake, glycolysis, pentose phosphate pathway, and oxidative phosphorylation. We did not identify significant differences in baseline cellular metabolism between control and KLF4-expressing cells. However, when mitochondrial function was impaired, KLF4 significantly increased spare respiratory capacity and levels of reactive oxygen species in the cells. To identify the biological effects of these changes, we analyzed proliferation and survival of control and KLF4-expressing cells under stress conditions, including serum and nutrition deprivation. We found that following serum starvation, KLF4 altered cell cycle progression by arresting the cells at the G2/M phase and that KLF4 protected cells from nutrition deprivation-induced death. Finally, we demonstrated that methylation-dependent KLF4-binding activity mediates mitochondrial fusion. Specifically, the downstream targets of KLF4-mCpG binding, guanine nucleotide exchange factors, serve as the effector of KLF4-induced mitochondrial fusion, cell cycle arrest, and cell protection. Our experimental system provides a robust model for studying the interactions between mitochondrial morphology and function, mitochondrial dynamics and metabolism, and mitochondrial fusion and cell death during tumor initiation and progression.

MRI tracking of autologous pancreatic progenitor-derived insulin-producing cells in monkeys.

Insulin-producing cells (IPCs) derived from a patient's own stem cells offer great potential for autologous transplantation in diabetic patients. However, the limited survival of engrafted cells remains a bottleneck in the application of this strategy. The present study aimed to investigate whether nanoparticle-based magnetic resonance (MR) tracking can be used to detect the loss of grafted stem cell-derived IPCs in a sensitive and timely manner in a diabetic monkey model. Pancreatic progenitor cells (PPCs) were isolated from diabetic monkeys and labeled with superparamagnetic iron oxide nanoparticles (SPIONs). The SPION-labeled cells presented as hypointense signals on MR imaging (MRI). The labeling procedure did not affect the viability or IPC differentiation of PPCs. Importantly, the total area of the hypointense signal caused by SPION-labeled IPCs on liver MRI decreased before the decline in C-peptide levels after autotransplantation. Histological analysis revealed no detectable immune response to the grafts and many surviving insulin- and Prussian blue-positive cell clusters on liver sections at one year post-transplantation. Collectively, this study demonstrates that SPIO nanoparticles can be used to label stem cells for noninvasive, sensitive, longitudinal monitoring of stem cell-derived IPCs in large animal models using a conventional MR imager.

Human induced pluripotent stem cell-derived neurons improve motor asymmetry in a 6-hydroxydopamine-induced rat model of Parkinson's disease.

BACKGROUND AIMS: Since human embryonic stem cells and human fetal neural stem cells have immune rejection and ethical issues, recent advancements in induced pluripotent stem cells (iPS cells) provide new possibilities to study autologous cell therapy for Parkinson's disease (PD). METHODS: We isolated human skin fibroblasts from normal individuals and patients with PD; we generated iPS cells by transfecting these human skin fibroblasts with retroviral reprogramming factors of OCT4, SOX2, KLF4 and c-MYC and induced iPS cells to differentiate neural stem cells (NSCs) and then into neurons and dopamine neurons in vitro. RESULTS: We found that iPS cell-derived NSC transplant into the striatum of the 6-hydroxydopamine (OHDA)-induced PD rats improved their functional defects of rotational asymmetry at 4, 8, 12 and 16 weeks after transplantation. iPS cell-derived NSCs were found to survive and integrate into the brain of transplanted PD rats and differentiated into neurons, including dopamine neurons in vivo. CONCLUSIONS: Transplantation of iPS cell-derived NSCs has therapeutic potential for PD. Our study provided experimental proof for future clinical application of iPS cells in cell-based treatment of PD.

Effects of age and sex on the hematology and blood chemistry of Tibetan macaques (Macaca thibetana).

Tibetan macaques (Macaca thibetana), also known as Chinese stump-tailed macaques, are a threatened primate species. Although Tibetan macaques are Old World monkeys in the genus of Macaca, limited age- and sex-related physiologic data are available for this particular species. We used 69 apparently healthy Tibetan male and female macaques to explore the effect of age and sex on physiologic parameters. Somatometric measurements, biochemistry, and hematologic parameters were analyzed. Significant age-related differences were found for weight, BMI, RBC count, Hgb, Hct, neutrophils, eosinophil count, ALT, AST, ALP, GGT, creatine kinase (muscle and brain subtypes), LDH, α-amylase, creatinine, apolipoprotein A1, total protein, albumin, cholesterol, HDL, and potassium. Significant differences by sex were noted for weight, BMI, ALT, total bilirubin, and indirect bilirubin. An interaction between age and sex accounted for statistically significant differences in the values for weight, BMI, and lymphocyte and eosinophil counts. These physiologic data will provide veterinarians and researchers with important age- and sex-specific reference ranges for evaluating experimental results from Tibetan macaques.

A preliminary report on oral fat tolerance test in rhesus monkeys.

BACKGROUND: Oral fat tolerance test (OFTT) has been widely used to assess the postprandial lipemia in human beings, but there is few studies concerning OFTT in nonhuman primates. This study is designed to explore the feasibility of OFTT in rhesus monkeys. METHODS: In a cross-over study, a total of 8 adult female rhesus monkeys were fed with normal monkey diet (NND), high sugar high fat diet (HHD), and extremely high fat diet (EHD), respectively. Each monkey consumed NND, HHD and EHD respectively, each weighing 60 g. Serial blood samples were collected at 1, 2, 3, 4, 5, and 6 h after ingesting each kind of food. Triglyceride, cholesterol, glucose, and insulin at each time point were measured. The area under the curve of triglyceride (TG-AUC) and triglyceride peak response (TG-PR) were also calculated. RESULTS: All monkeys ingested 3 kinds of foods within 15 minutes. TG-AUC and TG-PR of HHD group were higher than those of the other two groups. Postprandial triglyceride levels at 2, 3, 4, and 5 hours in HHD group during OFTT were also higher than those in NND and EHD group. CONCLUSIONS: HHD diet can be used in OFTT for nonhuman primates.

Function of mouse embryonic stem cell-derived supporting cells in neural progenitor cell maturation and long term expansion.

BACKGROUND: In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact. CONCLUSIONS/SIGNIFICANCE: The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.

Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

Labeling of cynomolgus monkey bone marrow-derived mesenchymal stem cells for cell tracking by multimodality imaging.

Recently, transplantation of allogeneic and autologous cells has been used for regenerative medicine. A critical issue is monitoring migration and homing of transplanted cells, as well as engraftment efficiency and functional capability in vivo. Monitoring of superparamagnetic iron oxide (SPIO) particles by magnetic resonance imaging (MRI) has been used in animal models and clinical settings to track labeled cells. A major limitation of MRI is that the signals do not show biological characteristics of transplanted cells in vivo. Bone marrow mesenchymal stem cells (MSCs) have been extensively investigated for their various therapeutic properties, and exhibit the potential to differentiate into cells of diverse lineages. In this study, cynomolgus monkey MSCs (cMSCs) were labeled with Molday ION Rhodamine-B™ (MIRB), a new SPIO agent, to investigate and characterize the biophysical and MRI properties of labeled cMSCs in vitro and in vivo. The results indicate that MIRB is biocompatible and useful for cMSCs labeling and cell tracking by multimodality imaging. Our method is helpful for detection of transplanted stem cells in vivo, which is required for understanding mechanisms of cell therapy.

17ß-estradiol protects dopaminergic neurons in organotypic slice of mesencephalon by MAPK-mediated activation of anti-apoptosis gene BCL2.

Both clinical and experimental studies provide growing evidences that marked sex differences in certain neurological disorders or disease models are largely attributed to the neuroprotective effects of estrogen. The purposes of this study were to assess the neuroprotective effect of 17ß-estradiol (E2) on dopaminergic neurons against 6-hydroxydopamine (6-OHDA) in organotypic mesencephalic slice culture and to elucidate the possible mechanism underlying neuroprotection. It was found that long-term exposure to E2 exerted marked effects on restoring the number of dopaminergic neurons, maintaining normal morphology of dopaminergic neurons, and preserving their ability to release dopamine at the presence of 6-OHDA. The neuroprotective effect of E2 could be dramatically blocked by an estrogen receptor antagonist ICI 182, 780 (ICI). The expression of GFAP, TLR4, and anti-apoptosis gene BCL2 were elevated at the presence of E2, whereas only BCL2 activation was blocked by ICI, dominantly responsible for E2-induced neuroprotection. Furthermore, activation of BCL2 was speculated to be mainly mediated through mitogen-activated protein kinase (MAPK) pathways, yet phosphatidylinositol-3-kinase signaling contributed largely to GFAP and TLR4 upregulation. Taken together, MAPK pathway-mediated BCL2 expression accounted for one of the key mechanisms involved in E2 neuroprotective effect on dopaminergic neurons against 6-OHDA insult. This finding provides new insight into controversial estrogen replacement therapy.

Insulin-producing cells from human pancreatic islet-derived progenitor cells following transplantation in mice.

Stem/progenitor cells hold promise for alleviating/curing type 1 diabetes due to the capacity to differentiate into functional insulin-producing cells. The current study aims to assess the differentiation potential of human pancreatic IPCs (islet-derived progenitor cells). IPCs were derived from four human donors and subjected to more than 2000-fold expansion before turning into ICCs (islet-like cell clusters). The ICCs expressed ISL-1 Glut2, PDX-1, ngn3, insulin, glucagon and somatostatin at the mRNA level and stained positive for insulin and glucagon by immunofluorescence. Following glucose challenge in vitro, C-peptide was detected in the sonicated ICCs, instead of in the conditioned medium. To examine the function of the cells in vivo, IPCs or ICCs were transplanted under the renal capsule of immunodeficient mice. One month later, 19 of 28 mice transplanted with ICCs and 4 of 14 mice with IPCs produced human C-peptide detectable in blood, indicating that the in vivo environment further facilitated the maturation of ICCs. However, among the hormone-positive mice, only 9 of 19 mice with ICCs and two of four mice with IPCs were able to secrete C-peptide in response to glucose.

Derivation and long-term culture of human parthenogenetic embryonic stem cells using human foreskin feeders.

PURPOSE: Feeder cells from animals raise considerable concern for contamination because they are directly in contact with embryonic stem cells. METHODS: To address this issue we collected discarded foreskin tissue and prepared a fibroblast cell line. We transferred one parthenogenetic blastocyst on to these feeder cells, and later observed outgrowth. By this approach, we were able to derive a human parthenogenetic embryonic stem cell line successfully. RESULTS: The embryonic stem cells had normal morphology, expressed all expected cell surface markers, could differentiate to embryonic bodies upon culture in vitro, and differentiated further to derivatives of all three germ layers. CONCLUSION: This study indicates that homologous human fibroblasts can be used as feeder cells to support not only the propagation, but also the derivation of ES cells, and this should facilitate studies of therapeutic cloning for research and clinical applications.

Suprachiasmatic nucleus slices induce molecular oscillations in fibroblasts.

The mammalian circadian pacemaker has been localized to the hypothalamic suprachiasmatic nucleus (SCN), where a set of clock genes and their resulting proteins form interlocking transcriptional/translational feedback loops to sustain molecular and functional oscillations. Interestingly, peripheral tissues and stimulated fibroblasts have also displayed daily oscillations, which are thought to be synchronized by the SCN in vivo. However, intercellular communications between the SCN and other tissues or cells remain poorly understood. Therefore, a novel co-culture model was established in the present study to understand the interactions between central and peripheral oscillators in co-cultures of SCN slices and NIH/3T3 cells in a serum-free condition. Expression profiles of Per1 and Rev-Erb alpha were measured in NIH/3T3 cells using real-time PCR. Results demonstrated that diffusible signals released from SCN slices could regulate molecular rhythms in cultured fibroblasts. Moreover, Rev-Erb alpha oscillation was more robust and appeared earlier than Per1.

Effect of basic fibroblast growth factor on the proliferation, migration and phenotypic modulation of airway smooth muscle cells.

BACKGROUND: Proliferation, cell migration and phenotypic modulation of airway smooth muscle cells (ASMCs) are important features of airway remodelling in asthma. The precise cellular and molecular mechanisms that regulate ASMCs proliferation, migration and phenotypic modulation in the lung remain unknown. Basic fibroblast growth factor (bFGF), a highly specific chemotactic and mitogenic factor for many cell types, appears to be involved in the development of airway remodelling. Our study assessed whether bFGF directly stimulates the proliferation, migration and phenotypic modulation of ASMCs. METHODS: Confluent and growth arrested human ASMCs were treated with human recombinant FGF. Proliferation was measured by BrdU incorporation and cell counting. Migration was examined using Boyden chamber apparatus. Expressions of smooth muscle (sm)-alpha-actin and sm-myosin heavy chain (MHC) isoform 1 were determined by RT-PCR and Western blot analysis. RESULTS: It was found that hrbFGF (10 ng/ml), when added to ASMCs, induced a significant increase in BrdU uptake and cell number by ASMCs as compared to controls and a significant increase in ASMCs migration with respect to controls. The mRNA and protein expressions of sm-alpha-actin and sm-MHC in ASMCs that were stimulated with hrbFGF decreased with respect to controls. CONCLUSION: It appears that bFGF can directly stimulate proliferation and migration of ASMCs, however, the expressions of cells' contractive phenotype decreased.

Inhibition of tyrosine hydroxylase expression in alpha-synuclein-transfected dopaminergic neuronal cells.

Although the aberrant expression of alpha-synuclein (alpha-Syn) is toxic to dopaminergic neurons, little is known about the correlation between the abnormality of alpha-Syn and the expression of tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of dopamine neurotransmitter. In this study, the MES23.5 rat dopaminergic cell line transfected with wild-type human alpha-Syn cDNA (h alpha-Syn) construct was used to investigate the association between alpha-Syn and TH. Immunocytochemical staining and Western blot for TH showed that the TH expression was greatly decreased in h alpha-Syn-transfected cells. Northern blot confirmed that the TH mRNA level was also dramatically reduced. Reduction of TH protein levels did not affect the growth and proliferation of the transfected cells. No apparent cell injury or death was observed. These results suggest that an abnormal expression of alpha-Syn may inhibit TH synthesis in dopaminergic cells.