Maneesha S. Inamdar

Harnessing the mesenchymal stem cell secretome for the treatment of cardiovascular disease.

The broad repertoire of secreted trophic and immunomodulatory cytokines produced by mesenchymal stem cells (MSCs), generally referred to as the MSC secretome, has considerable potential for the treatment of cardiovascular disease. However, harnessing this MSC secretome for meaningful therapeutic outcomes is challenging due to the limited control of cytokine production following their transplantation. This review outlines the current understanding of the MSC secretome as a therapeutic for treatment of ischemic heart disease. We discuss ongoing investigative directions aimed at improving cellular activity and characterizing the secretome and its regulation in greater detail. Finally, we provide insights on and perspectives for future development of the MSC secretome as a therapeutic tool.

Derivation and characterization of two sibling human embryonic stem cell lines from discarded grade III embryos.

Human embryonic stem (hES) cells are a valuable tool for studying human development in addition to their potential applications in regenerative medicine and drug discovery. The role of genetic background and epigenetic influences in development as well as in response to external influences such as drugs and therapies is well recognized. The great ethnic diversity in the Indian subcontinent translates to interindividual variability in drug response and disease susceptibility. For these reasons, new hES cell lines representing Indian genetic diversity will be valuable in studies of tissue-differentiation, cellular-function and for aspects of characterization of responses to drugs. We have derived two new hES cell lines, BJNhem19 and BJNhem20 from the inner cell mass (ICM) of discarded grade III human embryos that were not suitable for in vitro fertility treatment. Human leukocyte antigen (HLA) isotype analysis shows that they are genetically distinct from existing hES cell lines. Short tandem repeat (STR) analysis shows that the two cell lines are derived from sibling embryos. These cell lines show an undifferentiated phenotype in culture for more than 65 passages, show normal karyotype and express pluripotency markers such as TRA-1-60, TRA-1-81, stage-specific embryonic antigen-4 (SSEA-4), alkaline phosphatase, DNMT3B, GABRB3, GDF3, OCT4, NANOG, SOX2, TERF1, TDGF, LEFTA, THY1, and REX1. While both cell lines can differentiate into derivatives of all three germ layers in vitro, only BJNhem20 can form teratomas when transplanted into mice. We observe an increased frequency of cardiomyocyte differentiation from BJNhem20 embryoid bodies in feeder-free cultures upon induction with DMSO. Cardiomyocytes purified from such cultures survive and show rhythmic contractions for several weeks in culture. These hES cell lines have been accepted for deposit in the U.K. Stem Cell Bank and will be a useful resource for the international stem cell community.

Post-embryonic pericardial cells of Drosophila are required for overcoming toxic stress but not for cardiac function or adult development

The Drosophila heart is composed of two cell types: cardioblasts (CB) and pericardial cells (PC). Whereas CBs act to maintain rhythmic contractions, the functions of accessory PCs are not clear. The close association between these two cell types has led to speculation of a cardio-regulatory role for PCs. However, we find that viability and cardiac function are normal in larvae following post-embryonic ablation of PCs by induced cell death. Removal of PCs during the larval instars or before metamorphosis results in viable and fertile adults. Interestingly, such animals have a reduced lifespan and increased sensitivity to toxic chemicals. Thus, although PCs may have an embryonic role in cardiogenesis, they do not appear to play a part later in cardiac function as suggested. However, the role of PCs in the uptake and sequestering of toxins, their sensitivity to toxic stress and the decreased lifespan of animals without PCs indicate the importance of PCs in organismal homeostasis.

The Drosophila Homolog of the Human Transcription Factor TEF-1, Scalloped, is Essential for Normal Taste Behavior

The scalloped (sd) locus of Drosophila melanogaster encodes a protein with a novel DNA binding domain bearing a high degree of similarity to the human transcription factor TEF-1 (Campbell et al., 1992). We demonstrate that sd mutants show defects in response to a number of taste stimuli. Higher stimulus concentrations are required to elicit behavioral responses from mutant larvae and adult flies. The electrophysiological responses of the peripheral taste neurons in the labellum were found to be normal, suggesting that an inability to detect stimuli is not the cause of the mutant phenotype. The range of mutant responses of sd alleles to salt and sugar stimuli define a functional requirement for the gene in the nervous system and provide an assay for the genetic and molecular analysis of this role.

Macromolecular uptake in Drosophila pericardial cells requires rudhira function

The vertebrate reticuloendothelial system (RES) functions to remove potentially damaging macromolecules, such as excess hormones, immune-peptides and -complexes, bacterial-endotoxins, microorganisms and tumor cells. Insect hemocytes and nephrocytes - which include pericardial cells (PCs) and garland cells - are thought to be functionally equivalent to the RES. Although the ability of both vertebrate scavenger endothelial cells (SECs) and PCs to sequester colloidal and soluble macromolecules has been demonstrated the molecular mechanism of this function remains to be investigated. We report here the functional characterization of Drosophila larval PCs with important insights into their cellular uptake pathways. We demonstrate the nephrocyte function of PCs in live animals. We also develop and use live-cell assays to show that PCs take up soluble macromolecules in a Dynamin-dependent manner and colloids by a Dynamin-independent pathway. We had earlier identified Drosophila rudhira (Drudh) as a specific marker for PCs. Using RNAi mediated knock-down we show that Drudh regulates macropinocytic uptake in PCs. Our study establishes important functions for Drosophila PCs, describes methods to identify and study them, provides a genetic handle for further investigation of their role in maintaining homeostasis and demonstrates that they perform key subsets of the roles played by the vertebrate RES.

ARF1–GTP regulates Asrij to provide endocytic control of Drosophila blood cell homeostasis

Significance Ras small GTPases including ARFs act as molecular switches to modulate signaling pathways involved in hematopoiesis. Decreased guanine nucleotide exchange factor activity or increased GTPase-activating protein activation are associated with many leukemias, myelodysplastic syndromes, and myeloproliferative disorders. Drosophila is a good model to address questions related to aberrant hematopoiesis. Using Drosophila genetics and gene expression analysis we show tissue-specific function of the ubiquitously expressed endocytic protein, Drosophila ARF1 by interaction of ARF1–GTP with a blood-cell–expressed endocytic protein Asrij. The ARF1–Asrij axis brings about endosomal regulation of multiple signaling pathways in hematopoiesis. Drosophila melanogaster larval hematopoiesis is a well-established model to study mechanisms that regulate hematopoietic niche maintenance and control of blood cell precursor (prohemocyte) differentiation. Molecules that perturb niche function affect the balance between prohemocytes and differentiated hemocytes. The conserved hemocyte-specific endosomal protein Asrij is essential for niche function and prohemocyte maintenance. Elucidating how subcellular trafficking molecules can regulate signaling presents an important challenge. Here we show that Asrij function is mediated by the Ras family GTPase Arf79F, the Drosophila homolog of ADP ribosylation factor 1 (ARF1), essential for clathrin coat assembly, Golgi architecture, and vesicular trafficking. ARF1 is expressed in the larval lymph gland and in circulating hemocytes and interacts with Asrij. ARF1-depleted lymph glands show loss of niche cells and prohemocyte maintenance with increased differentiation. Inhibiting ARF1 activation by knocking down its guanine nucleotide exchange factor (Gartenzwerg) or overexpressing its GTPAse-activating protein showed that ARF1–GTP is essential for regulating niche size and maintaining stemness. Activated ARF1 regulates Asrij levels in blood cells thereby mediating Asrij function. Asrij controls crystal cell differentiation by affecting Notch trafficking. ARF1 perturbation also leads to aberrant Notch trafficking and the Notch intracellular domain is stalled in sorting endosomes. Thus, ARF1 can regulate Drosophila blood cell homeostasis by regulating Asrij endocytic function. ARF1 also regulates signals arising from the niche and differentiated cells by integrating the insulin-mediated and PDGF-VEGF receptor signaling pathways. We propose that the conserved ARF1–Asrij endocytic axis modulates signals that govern hematopoietic development. Thus, Asrij affords tissue-specific control of global mechanisms involved in molecular traffic.

Asrij maintains the stem cell niche and controls differentiation during Drosophila lymph gland hematopoiesis.

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation.

Human BCAS3 Expression in Embryonic Stem Cells and Vascular Precursors Suggests a Role in Human Embryogenesis and Tumor Angiogenesis

Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3) is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES) cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker.

Conserved Regulation of the JAK/STAT Pathway by the Endosomal Protein Asrij Maintains Stem Cell Potency

SUMMARY Asrij/OCIAD1 is an endosomal protein expressed in stem cells and cardiovascular lineages and aberrantly expressed in several cancers. We show that dose-dependent modulation of cytokine-dependent JAK/STAT signaling by Asrij regulates mouse embryonic stem cell pluripotency as well as Drosophila hematopoietic stem cell maintenance. Furthermore, mouse asrij can substitute for Drosophila asrij, indicating that they are true homologs. We identify a conserved region of Asrij that is necessary and sufficient for vesicular localization and function. We also show that Asrij and STAT3 colocalize in endosomes and interact biochemically. We propose that Asrij provides an endosomal scaffold for STAT3 interaction and activation, and may similarly control other circuits that maintain stemness. Thus, Asrij provides a key point of control for spatial and kinetic regulation of stem cell signals.

Global Solutions to the Challenges of Setting up and Managing a Stem Cell Laboratory

Keywords Stem cell laboratory establishment.Laboratorydesign.Laboratory operation.Good cell culture practice.Laboratory management.Standardization.Quality controlBackground and OriginIn recent years, stem cell research has made major contribu-tions to our understanding of biology. The ability toreprogramme somatic cells to stem cells of desired potencyhas made this exciting area of research accessible to almostevery laboratory, in spite of varying ethical, political andeconomic scenarios. The promise of stem cell research inmaking regenerative medicine accessible has further attractedclinicians, materials scientists, chemists, physicists and othernon-specialists to this field of research. While this situation isdesirable, the novice usually faces the daunting task of settingupandmaintainingastemcelllaboratory,oftenwithoutaccessto local expertise.Researchers allocate a significantamount ofresource to keep their approach technologically advanced.Deliveryofrobustandreliabledata(i.e.achievingreliableandreproducible stem cell cultures for experimentation) is oftenneglected.Thiscanresultindisruptionanddelayinlaboratorywork and at worst, wasted research resource and evenretractionofpublications.Thisdocumentlaysoutfundamentalissues to be addressed in the establishment of a stem cellculture laboratory. The aim is to provide guidance on ways toovercome many challenges to smooth operation, encounteredin varying climates and environments. Parts of this overvieware modeled on the Guidance on Good Cell Culture Practice[3] and should be considered as an ‘aide memoire’ tocomplement existing guidelines. This guidance originatedfrom experience gained by the authors in the establishment ofmultiple cell culture laboratories and training students indifferent countries with widely different environmentalconditions in Northern Europe and in India.ScopeThis document addresses the full range of issues that new aswell as established stem cell researchers charged with settingup a stem cell laboratory may face. It proposes solutions todealwithpotentialproblemsaheadoftime.Theaimistohelpincrease reproducibility of procedures, reduceuncertainties insupply, and help academics meet international scientific andethical requirements.IntroductionGood scientific practice and maintenance of high standardsof mammalian cell culture is important for any researchbased on the use of stem cell lines. Contributions to stemcell research are now global and include researchers andcountries that are relatively new to the field. One importantgoal is to establish consistent standards of scientific andtechnical competence in stem cell culture that will promotegood science and efficient use of research resources. Thispaper identifies generic guidance for establishment and