Kuldip S. Sidhu

Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.

Human embryonic stem cells (hESCs) are potential renewable sources of cells in replacement therapies for many diseases including type 1 diabetes. We have established a three dimensional (3D) model to culture and differentiate hESCs that are encapsulated in calcium alginate microcapsules. This system promotes cellular interactions that are essential for both maintaining pluripotency and differentiation. This 3D model also provides opportunity to separate out hESCs from fibroblasts used as feeder layer during culture. In this study, we compared the viability and proliferation of the encapsulated hESCs cultured in serum replacement (SR) medium, human fetal fibroblast-conditioned medium (hFF-CM), in the presence and absence of Y-27632, a ROCK inhibitor. Treatment of hESCs with Y-27632 promoted cell survival, cell cluster formation and proliferation rate in both SR medium and hFF-CM. These encapsulated hESC clusters were then directly differentiated to definitive endoderm cells that expressed mesendoderm (Brachyury 70-fold), definitive endoderm (SOX17>300-fold, FOXA2>800-fold, and CXCR4>100-fold) and primitive gut tube (HNF1beta>120-fold) as compared to the undifferentiated hESCs. These data show that microcapsules can be used for differentiation of hESCs into definitive endoderm in 3D and could have potential application for immune-isolation and prevention of teratomas formation of hESCs during transplantation.

Growth and Differentiation of Embryoid Bodies Derived from Human Embryonic Stem Cells: Effect of Glucose and Basic Fibroblast Growth Factor

Abstract Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.

Differentiation of encapsulated embryonic stem cells after transplantation.

Background. Embryonic stem cells (ESC) when transplanted into recipients with different major histocompatibility antigens may be rejected, especially as cells differentiate and expression of these antigens increases. One method to prevent rejection is to place the developing ESC in microcapsules. It is currently unknown what effect encapsulation has on the ability of ESC to differentiate. Methods. Human ESC (hESC; hES03 line) and mouse ESC (mESC; R1 line) were encapsulated in 2.2% barium alginate and transplanted intraperitoneally in SCID and BALB/c mice respectively. Cell morphology, viability, and gene characterization were assessed after retrieving the capsules up to four weeks from SCID mice and three months from BALB/c mice. Results. Encapsulation prevented hESC and mESC from forming teratomas up to four weeks and three months, respectively. mESC but not hESC formed aggregates within the capsules, which remained free of fibrosis. Some but not all the transplanted encapsulated hESC differentiated towards all three lineages, but more so towards an endodermal lineage as shown by increased expression of alpha fetoprotein. This was similar to what occurred when encapsulated and non-encapsulated hESC were cultured in vitro for two weeks. In contrast to the hESC, transplanted encapsulated mESC differentiated mostly towards an ectodermal lineage as shown by increased expression of nestin and glial fibrillary acidic protein. In vitro, encapsulated and nonencapsulated mESC also began to differentiate, but not down any specific lineage. Conclusions. Encapsulated ESC do differentiate, although along multiple pathways, both when transplanted and maintained in culture, just as nonencapsulated ESC do when removed from their feeder layer.

Derivation of male germ cell-like lineage from human fetal bone marrow stem cells

Mesenchymal stem cells derived from bone marrow are a well characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. Reports have shown that murine and human embryonic stem cells can differentiate into primordial germ cells and then to early gametes. Evidence has indicated that some adult stem cells also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of human mesenchymal stem cells into germ cells. This study investigated the ability of retinoic acid and testicular extracts to induce human bone marrow stem cells (hBMSC) to differentiate into male germ cells. It was found that a small population of hBMSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers OCT4, STELLA, NANOG and VASA, and male germ-ceil-specific markers such as DAZL, TH2, c-kit, beta(1)-integrin, ACR, PRMl, FSHR, STRA8 and SCP3, as analysed by reverse transcription-polymerase chain reaction and immunohistochemistry. These results demonstrated that hBMSC may differentiate into male germ cells and the same could be used as a potential source of cells for reproductive toxicological studies.

A Combined Epigenetic and Non-Genetic Approach for Reprogramming Human Somatic Cells

Reprogramming of somatic cells to different extents has been reported using different methods. However, this is normally accompanied by the use of exogenous materials, and the overall reprogramming efficiency has been low. Chemicals and small molecules have been used to improve the reprogramming process during somatic cell nuclear transfer (SCNT) and induced pluripotent stem (iPS) cell generation. We report here the first application of a combined epigenetic and non-genetic approach for reprogramming somatic cells, i.e., DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and human embryonic stem cell (hESC) extracts. When somatic cells were pretreated with these inhibitors before exposure to hESC (MEL1) extracts, morphological analysis revealed a higher rate of hESC-like colony formation than without pretreatment. Quantitative PCR (qPCR) demonstrated that pluripotency genes were upregulated when compared to those of somatic cells or treated with hESC extracts alone. Overall changes in methylation and acetylation levels of pretreated somatic cells suggests that epigenetic states of the cells have an effect on reprogramming efficiency induced by hESC extracts. KnockOutserum replacement (KOSR™) medium (KO-SR) played a positive role in inducing expression of the pluripotency genes. hESC extracts could be an alternative approach to reprogram somatic cells without introducing exogenous materials. The epigenetic pre-treatment of somatic cells could be used to improve the efficiency of reprogramming process. Under differentiation conditions, the reprogrammed cells exhibited differentiation ability into neurons suggesting that, although fully reprogramming was not achieved, the cells could be transdifferentiated after reprogramming.

Recent Advances in the Derivation of Germ Cells from the Embryonic Stem Cells

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.

New approaches for the generation of induced pluripotent stem cells

Introduction: The advent of induced pluripotent stem cell (iPSC) technology has opened up new vistas to generate patient-specific pluripotent stem cells from somatic cells. During the last 5 years, the iPSCs produced from a variety of somatic cell sources are found to be very similar, if not identical to embryonic stem cells. Invariably these cells are produced by viral transduction of four transcriptional factors that renders these cells unfit for therapeutic purposes. Areas covered: This review discusses current developments emphasising on new and improved methods of generating iPSCs, including minimal or no genetic modifications via excisable lentiviral and transposon vectors or through repeated application of transient plasmid, episomal and adenovirus vectors. Recent use of small molecules, synthetic mRNA and microRNAs is also reviewed. Expert opinion: iPSC technology is emerging as an unprecedented opportunity in biomedical research, disease modeling, drug discovery and regenerative medicine. However, to harness the full potential of this technology, a number of issues that need to be resolved pertaining to iPSC safety, stability, culture variability, their comparison with ES cells, the reprogramming mechanisms and better ways to direct a specific reprogramming process including lineage specifications.

Induced pluripotent stem cells as tools for disease modelling and drug discovery in Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative brain disorder that leads to a progressive decline in a person’s memory and ability to communicate and carry out daily activities. The brain pathology in AD is characterized by extensive neuronal loss, particularly of cholinergic neurons, intracellular neurofibrillary tangles composed of the tau protein (NFTs) and extracellular deposition of plaques composed of β-amyloid (Aβ), a cleavage product of the amyloid precursor protein (APP). These two insoluble protein aggregates are accompanied by a chronic inflammatory response and extensive oxidative damage. Whereas dys-regulation of APP expression or processing appears to be important for the familial, early-onset form of AD, controversy exists between the “Baptists” (in favour of Aβ) and the “Tauists” (in favour of tau) as to which of these two protein dysfunctions occur at the earliest stages or are the most important contributors to the disease process in sporadic AD. However, more and more “non-amyloid” and “non-tau” causes have been proposed, including, glycation, inflammation, oxidative stress and dys-regulation of the cell cycle. However, to get an insight into the ultimate cause of AD, and to prove that any drug target is valuable in AD, disease-relevant models giving insight into the pathogenic processes in AD are urgently needed. In the absence of a good animal model for sporadic AD, we propose in this review that induced pluripotent stem cells, derived from dermal fibroblasts of AD patients, and differentiated into cholinergic neurons, might be a promising novel tool for disease modelling and drug discovery for the sporadic form of AD.

Age-specific effects of voluntary exercise on memory and the older brain.

BACKGROUND Physical exercise in early adulthood and mid-life improves cognitive function and enhances brain plasticity, but the effects of commencing exercise in late adulthood are not well-understood. METHOD We investigated the effects of voluntary exercise in the restoration of place recognition memory in aged rats and examined hippocampal changes of synaptic density and neurogenesis. RESULTS We found a highly selective age-related deficit in place recognition memory that is stable across retest sessions and correlates strongly with loss of hippocampal synapses. Additionally, 12 weeks of voluntary running at 20 months of age removed the deficit in the hippocampally dependent place recognition memory. Voluntary running restored presynaptic density in the dentate gyrus and CA3 hippocampal subregions in aged rats to levels beyond those observed in younger animals, in which exercise had no functional or synaptic effects. By contrast, hippocampal neurogenesis, a possible memory-related mechanism, increased in both young and aged rats after physical exercise but was not linked with performance in the place recognition task. We used graph-based network analysis based on synaptic covariance patterns to characterize efficient intrahippocampal connectivity. This analysis revealed that voluntary running completely reverses the profound degradation of hippocampal network efficiency that accompanies sedentary aging. Furthermore, at an individual animal level, both overall hippocampal presynaptic density and subregional connectivity independently contribute to prediction of successful place recognition memory performance. CONCLUSIONS Our findings emphasize the unique synaptic effects of exercise on the aged brain and their specific relevance to a hippocampally based memory system for place recognition.

Derivation of Motor Neurons from three Clonal Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESC) demonstrate a remarkable proliferative and developmental potential and thus have huge therapeutic potential. To direct the differentiation of hESC to a specific lineage of high purity for cell transplantation is highly desirable. Here we describe a modified in vitro procedure to direct differentiation of three clonal hESC lines, hES 3.1, hES 3.2 and hES 3.3 efficiently to spinal motor neurons by using various differentiation factors namely retinoic acid (RA), sonic hedgehog (Shh), bone morphogenetic protein-2 (BMP-2) and Wnt3A. The highest number of motor neurons (58.0 +/- 7.6%) were obtained by an early treatment of embryoid bodies with a combination of RA + Shh from all the clonal hESC lines combined. The hES 3.1 line, however, produced relatively more motor neurons (69.5 +/- 11.8%) compared to other two hES clones, 3.2 (52.4 +/- 13.1%) and 3.3 (52.3 +/- 15.5%). Immunolocalisation studies revealed the expression of neuronal specific marker, beta omega-tubulin and motor neuron specific marker, HB9/HLXB9 in all the three hESC clones after 45 days of differentiation. The RT-PCR analyses showed the presence of the neuron-specific genes. This modified differentiation protocol provides a mean of obtaining an enriched population of motor neurons from hESC for possible use in studies of lineage development, drug discovery and also as a potential cell therapy for motor neuron disease.