Desired Cell Types Research Articles

326-LB: BCL-xL/BCL2L1 Is a Critical Anti-Apoptotic Protein that Suppresses BAK to Promote Pancreatic Specification from Human Pluripotent Stem Cells

The differentiation of human pluripotent stem cells (hPSCs) into desired cell types such as the pancreatic cells involve cellular proliferation and apoptosis during cell fate transitions. Although different rates of cell growth and death are typically observed during differentiation, its implications for pancreatic tissue formation in humans remain unclear. Here, we profiled the expression of BCL-2 family of proteins during pancreatic specification from hPSCs and observed an upregulation of BCL- XL, downregulation of BAK and corresponding downregulation of cleaved CASP3 representative of apoptosis. The inhibition or downregulation of BCL-xL reciprocally increased apoptosis and resulted in a decreased gene expression of pancreatic markers despite a compensatory increase in companion anti-apoptotic protein BCL2. RNA-Seq analyses revealed a downregulation of multiple metabolic genes upon inhibition of BCL-xL function. Bioenergetics assays then revealed a broad downregulation of both glycolysis and oxidative phosphorylation when BCL-xL function was inhibited. In conclusion, we have identified anti-apoptotic protein BCL-xL to be necessary for suppressing the expression of pro-apoptotic protein BAK to facilitate proper pancreas formation in human cells. This is the first report that links a member of the BCL-2 family with pancreatic specification. Therefore, modulation of these two BCL-2 family of proteins can potentially increase the survival and robustness of pancreatic progenitors that ultimately defines human pancreatic beta cell mass and function. Disclosure L. Loo: None. A. Teo: None. S. Ghosh: None. A. Soetedjo: None. L. Nguyen: None. V.G. Krishnan: None. S. Hoon: None. Funding Agency for Science, Technology and Research; Institute of Molecular and Cell Biology

Generation of mesenchymal stem-like cells for producing extracellular vesicles

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with therapeutic potential against autoimmune diseases, inflammation, ischemia, and metabolic disorders. Contrary to the previous conceptions, recent studies have revealed that the tissue repair and immunomodulatory functions of MSCs are largely attributed to their secretome, rather than their potential to differentiate into desired cell types. The composition of MSC secretome encompasses cytokines and growth factors, in addition to the cell-derived structures known as extracellular vesicles (EVs). EVs are membrane-enclosed nanoparticles that are capable of delivering biomolecules, and it is now believed that MSC-derived EVs are the major players that induce biological changes in the target tissues. Based on these EVs’ characteristics, the potential of EVs derived from MSC (MSC-EV) in terms of tissue regeneration and immune modulation has grown during the last decade. However, the use of MSCs for producing sufficient amount of EVs has not been satisfactory due to limitations in the cell growth and large variations among the donor cell types. In this regard, pluripotent stem cells (PSCs)-derived MSC-like cells, which can be robustly induced and expanded in vitro, have emerged as more accessible cell source that can overcome current limitations of using MSCs for EV production. In this review, we have highlighted the methods of generating MSC-like cells from PSCs and their therapeutic outcome in preclinical studies. Finally, we have also discussed future requirements for making this cell-free therapy clinically feasible.

Enhanced Transfection of Human Mesenchymal Stem Cells Using a Hyaluronic Acid/Calcium Phosphate Hybrid Gene Delivery System

Human mesenchymal stem cells (hMSCs) show enormous potential in regenerative medicine and tissue engineering. However, current use of hMSCs in clinics is still limited because there is no appropriate way to control their behavior in vivo, such as differentiation to a desired cell type. Genetic modification may provide an opportunity to control the cells in an active manner. One of the major hurdles for genetic manipulation of hMSCs is the lack of an efficient and safe gene delivery system. Herein, biocompatible calcium phosphate (CaP)-based nanoparticles stabilized with a catechol-derivatized hyaluronic acid (dopa-HA) conjugate were used as a carrier for gene transfection to hMSCs for improved differentiation. Owing to the specific interactions between HA and CD44 of bone marrow-derived hMSCs, dopa-HA/CaP showed significantly higher transfection in hMSCs than branched polyethylenimine (bPEI, MW 25 kDa) with no cytotoxicity. The co-delivery of a plasmid DNA encoding bone morphogenetic protein 2 (BMP-2 pDNA) and micro RNA 148b (miRNA-148b) by dopa-HA/CaP achieved significantly improved osteogenic differentiation of hMSCs.

Serotonin Neuronal Function from the Bed to the Bench: Is This Really a Mirrored Way?

Induced pluripotent stem cells (iPSCs) offer a great opportunity to recapitulate both normal and pathologic development of brain tissues. Recently, three research teams have developed human-PSC technology and direct somatic cell reprogramming to allow induction of human serotonin (5-hydroxytryptamine; 5-HT) neurons in vitro . While preclinical studies have repeatedly shown that 5-HT suppresses 5-HT neuronal firing activity, one group has tested the effect of 5-HT on the neuronal activity of those 5-HT-like cells and found a paradoxical excitatory action of 5-HT. Here, we argue that few cautions in translational interpretations have to be taken into account. Nonetheless, using patient-derived cells for generating disease relevant cell types truly offers a new and powerful approach for investigating mechanisms playing fundamental roles in psychiatric disorders. Disease modeling by direct reprogramming into desired cell types represents a new huge challenge. Induced pluripotent stem cells (iPSCs), cells reprogrammed from human somatic cells, offer a great opportunity to recapitulate both normal and pathologic development of brain tissues and may as well provide essential strategies toward cell-based therapy of neuropsychiatric diseases (Vadodaria et al., 2018). Successfully, in 2016, three research teams have developed human-PSC technology (Lu et al., 2016) and direct somatic cell reprogramming (Vadodaria et al., 2016a; Xu et al., 2016) to allow induction of human serotonin neurons in vitro for the first time (for review, see Vadodaria et al., 2016b). Remarkably, Lu et al. (2016) have demonstrated the accurate timely regulation of WNT, SHH, and FGF4 signaling pathways during the serotonergic (5-HT) neuron differentiation and generated an enriched population of 5-HT neurons from human embryonic stem cells (ESCs) and iPSCs. These human …

WNT Inhibition and Increased FGF Signaling Promotes Derivation of Less Heterogeneous Primed Human Embryonic Stem Cells, Compatible with Differentiation.

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However, standard culture conditions maintain hESCs in a primed state, which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks, primed hESCs have been converted to a naive state, but this has restricted the efficiency of existing directed differentiation protocols. In mouse, WNT inhibition by inhibitor of WNT production-2, together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI), markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study, we show that DhiFI conditions similarly improved primed hESC traits, such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages, DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover, DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers, compared to primed-derived lines from the same PICMI. Overall, DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types.

Integrated Multi-Assay Culture Model for Stem Cell Chondrogenic Differentiation.

Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-β) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens.

3D2 Self‐Assembling Janus Peptide Dendrimers with Tailorable Supermultivalency

AbstractThe self‐assembly of peptides enables the construction of self‐assembled peptide nanostructures (SPNs) with chemical composition similar to those of natural proteins; however, the structural complexity and functional properties of SPNs are far beneath those of natural proteins. One of the most fundamental challenges in fabricating more elaborate SPNs lies in developing building blocks that are simultaneously more complex and relatively easy to synthesize. Here, the development of self‐assembling Janus peptide dendrimers (JPDs) is reported, which have fully 3D structures similar to those of globular proteins. For the reliable and convenient synthesis of JPDs, a solid‐phase bifurcation synthesis method is devised. The self‐assembly behavior of JPDs is unique because only the dendrimer generation and not the weight fraction dictates the morphology of SPNs. The coassembly of two JPD building blocks provides an opportunity not only to enlarge the morphological repertoire in a predictable manner but also to discover SPNs with unusual and interesting morphologies. Because JPD assemblies have dual multivalency, i.e., supramolecular and unimolecular multivalency, the JPD system enables the statistical selection of materials with high avidity for the desired cell types and possibly any target receptors.

Исследование функций мозга генетически кодируемыми инструментами

Methods based on genetically-encoded molecular constructs, such as antisense-knockdown and RNA interference that alter the expression of target genes, are widely used to analyze the function of proteins encoded by these genes, and also find application in medical practice. Using these methods, for example, we found that even a short-term decrease in the expression of one of the norepinephrine receptors during the critical period of brain development leaves a long-lasting impact on the neurochemical and behavioral traits in later life. Delivery into the brain cells of viral vectors encoding any proteins that affect cell function or small hairpin RNA (shRNA) that reduce the expression of the target gene, also finds use in neurobiology. A vivid manifestation of the power of genetically encoded instruments in studies of the central nervous system, instruments potentially suitable for controlling the activity of brain cells for therapeutic purposes are optogenetics and chemogenetics. Both approaches realized by expressing receptors in the desired cell type that are new to the body, reacting to light of a certain wavelength or a chemical ligand unusual for the body. These approaches make it possible to evaluate the functional consequences of changes in the activity of a specific population of neurons, which, for example, has provided significant progress in deciphering the mechanisms of central regulation of behavior. For example, using optogenetics, we found that the activation of glutamatergic neurons of the dorsal hippocampus induces a depressive-like behavior, and the antidepressant effect of ketamine on this behavior produced by its direct action on the NMDA receptors. Developed in the past few years, genome editing and gene expression management techniques based on bacterial CRISPR/Cas systems already used to study brain function. At present, possible applications of opto- and chemogenetics, as well as CRISPR/Cas technologies in medicine are being developed on model objects with optimistic expectations.

Brain-Derived Neurotrophic Factor in Central Nervous System Myelination: A New Mechanism to Promote Myelin Plasticity and Repair.

Brain-derived neurotrophic factor (BDNF) plays vitally important roles in neural development and plasticity in both health and disease. Recent studies using mutant mice to selectively manipulate BDNF signalling in desired cell types, in combination with animal models of demyelinating disease, have demonstrated that BDNF not only potentiates normal central nervous system myelination in development but enhances recovery after myelin injury. However, the precise mechanisms by which BDNF enhances myelination in development and repair are unclear. Here, we review some of the recent progress made in understanding the influence BDNF exerts upon the myelinating process during development and after injury, and discuss the cellular and molecular mechanisms underlying its effects. In doing so, we raise new questions for future research.

Methyl 3,4-Dihydroxybenzoate Induces Neural Stem Cells to Differentiate Into Cholinergic Neurons in vitro.

Neural stem cells (NSCs) have been shown as a potential source for replacing degenerated neurons in neurodegenerative diseases. However, the therapeutic potential of these cells is limited by the lack of effective methodologies for controlling their differentiation. Inducing endogenous pools of NSCs by small molecule can be considered as a potential approach of generating the desired cell types in large numbers. Here, we reported the characterization of a small molecule (Methyl 3,4-dihydroxybenzoate; MDHB) that selectively induces hippocampal NSCs to differentiate into cholinergic motor neurons which expressed synapsin 1 (SYN1) and postsynaptic density protein 95 (PSD-95). Studies on the mechanisms revealed that MDHB induced the hippocampal NSCs differentiation into cholinergic motor neurons by inhibiting AKT phosphorylation and activating autophosphorylation of GSK3β at tyrosine 216. Furthermore, we found that MDHB enhanced β-catenin degradation and abolished its entering into the nucleus. Collectively, this report provides the strong evidence that MDHB promotes NSCs differentiation into cholinergic motor neurons by enhancing gene Isl1 expression and inhibiting cell cycle progression. It may provide a basis for pharmacological effects of MDHB directed on NSCs.

GalNAc Conjugation Attenuates the Cytotoxicity of Antisense Oligonucleotide Drugs in Renal Tubular Cells

Targeted delivery of antisense oligonucleotide (AON) drugs is a promising strategy to increase their concentration in the desired tissues and cell types while reducing access to other organs. Conjugation of AONs to N-acetylgalactosamine (GalNAc) has been shown to efficiently shift their biodistribution toward the liver via high-affinity binding to the asialoglycoprotein receptor (ASGPR) expressed at the surface of hepatocytes. Nevertheless, GalNAc conjugation does not prevent accumulation of AONs in the kidney cortex, and GalNAc-conjugated AONs might cause kidney toxicities, for example, under conditions of ASGPR saturation. Here, we investigated the nephrotoxicity potential of GalNAc-conjugated AONs by in vitro profiling of AON libraries in renal proximal tubule epithelial cells (PTECs) and in vivo testing of selected candidates. Whereas GalNAc-conjugated AONs appeared generally innocuous to PTECs, some caused mild-to-moderate nephrotoxicity in rats. Interestingly, the in vivo kidney liabilities could be recapitulated in vitro by treating PTECs with the unconjugated (or naked) parental AONs. An in vitro mechanistic study revealed that GalNAc conjugation attenuated AON-induced renal cell toxicity despite intracellular accumulation similar to that of naked AONs and independent of target knockdown. Overall, our in vitro findings reveal ASGPR-independent properties of GalNAc AONs that confer a favorable safety profile at the cellular level, which may variably translate in vivo due to catabolic transformation of circulating AONs.

An Insight into DNA-free Reprogramming Approaches to Generate Integration-free Induced Pluripotent Stem Cells for Prospective Biomedical Applications.

More than a decade ago, a pioneering study reported generation of induced Pluripotent Stem Cells (iPSCs) by ectopic expression of a cocktail of reprogramming factors in fibroblasts. This study has revolutionized stem cell research and has garnered immense interest from the scientific community globally. iPSCs hold tremendous potential for understanding human developmental biology, disease modeling, drug screening and discovery, and personalized cell-based therapeutic applications. The seminal study identified Oct4, Sox2, Klf4 and c-Myc as a potent combination of genes to induce reprogramming. Subsequently, various reprogramming factors were identified by numerous groups. Most of these studies have used integrating viral vectors to overexpress reprogramming factors in somatic cells to derive iPSCs. However, these techniques restrict the clinical applicability of these cells as they may alter the genome due to random viral integration resulting in insertional mutagenesis and tumorigenicity. To circumvent this issue, alternative integration-free reprogramming approaches are continuously developed that eliminate the risk of genomic modifications and improve the prospects of iPSCs from lab to clinic. These methods establish that integration of transgenes into the genome is not essential to induce pluripotency in somatic cells. This review provides a comprehensive overview of the most promising DNA-free reprogramming techniques that have the potential to derive integration-free iPSCs without genomic manipulation, such as sendai virus, recombinant proteins, microRNAs, synthetic messenger RNA and small molecules. The understanding of these approaches shall pave a way for the generation of clinical-grade iPSCs. Subsequently, these iPSCs can be differentiated into desired cell type(s) for various biomedical applications.

SERPINA9 and SERPINB2: Novel Cartilage Lineage Differentiation Markers of Human Mesenchymal Stem Cells with Kartogenin.

Human mesenchymal stem cells (hMSCs) are a promising source for regenerative medicine, especially mesodermal lineages. Clinical applications require an understanding of the mechanisms for transcriptional control to maintain the desired cell type. The aim of this study was to identify novel markers for differentiation of hMSCs into bone or cartilage with the use of Kartogenin, by RNA analysis using microarray technology, and explore the role of RhoA-Rho associated protein kinase (ROCK) inhibition in these. Commercial human bone marrow derived primary mesenchymal stem cells were purchased from ATCC. Cells were differentiated in vitro in 2-dimensional cultures using Kartogenin as the main cartilage inducer and bone morphogenetic protein 2 for bone differentiation; cells were cultured with and without ROCK inhibitor Y-27632. After 21 days of culture, whole RNA was extracted and analyzed via Affimetrix microarrays. The most significant hits were validated by quantitative polymerase chain reaction. We found a total of 1,757 genes that were either up- or downregulated on differentiation, when compared to P1 hMSC (control) at day 0 of differentiation. Two members of the Serpin superfamily, SERPINA9 and SERPINB2, were significantly upregulated in the cartilage groups, whereas they were unchanged in the bone groups with and without ROCK inhibition. SERPINA9 and SERPINB2 are novel differentiation markers, and molecular regulator candidates for hMSC lineage commitment toward bone and cartilage, providing a new tool for regenerative medicine. Our study highlights the roles of these 2 genes, with significant upregulation of both in cell cultures stimulated with Kartogenin.

Who Will Win: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells for β Cell Replacement and Diabetes Disease Modeling?

Ever since the reprogramming of human fibroblasts to induced pluripotent stem cells (hiPSCs), scientists have been trying to determine if hiPSCs can give rise to progeny akin to native terminally differentiated cells as human embryonic stem cells (hESCs) do. Many different somatic cell types have been successfully reprogrammed via a variety of methods. In this review, we will discuss recent studies comparing hiPSCs and hESCs and their ability to differentiate to desired cell types as well as explore diabetes disease models. Both somatic cell origin and the reprogramming method are important to the epigenetic state of the hiPSCs; however, genetic background contributes the most to differences seen between hiPSCs and hESCs. Based on our review of the relevant literature, hiPSCs display differences compared to hESCs, including a higher propensity for specification toward particular cell types based on memory retained from the somatic cell of origin. Moreover, hiPSCs provide a unique opportunity for creating diabetes disease models.

Versatile targeting system for lentiviral vectors involving biotinylated targeting molecules

Conjugating certain types of lentiviral vectors with targeting ligands can redirect the vectors to specifically transduce desired cell types. However, extensive genetic and/or biochemical manipulations are required for conjugation, which hinders applications for targeting lentiviral vectors for broader research fields. We developed envelope proteins fused with biotin-binding molecules to conjugate the pseudotyped vectors with biotinylated targeting molecules by simply mixing them. The envelope proteins fused with the monomeric, but not tetrameric, biotin-binding molecules can pseudotype lentiviral vectors and be conjugated with biotinylated targeting ligands. The conjugation is stable enough to redirect lentiviral transduction in the presence of serum, indicating their potential in in vivo . When a signaling molecule is conjugated with the vector, the conjugation facilitates transduction and signaling in a receptor-specific manner. This simple method of ligand conjugation and ease of obtaining various types of biotinylated ligands will make targeted lentiviral transduction easily applicable to broad fields of research.

Revisiting the Advances in Isolation, Characterization and Secretome of Adipose-Derived Stromal/Stem Cells.

Adipose-derived stromal/stem cells (ASCs) seems to be a promising regenerative therapeutic agent due to the minimally invasive approach of their harvest and multi-lineage differentiation potential. The harvested adipose tissues are further digested to extract stromal vascular fraction (SVF), which is cultured, and the anchorage-dependent cells are isolated in order to characterize their stemness, surface markers, and multi-differentiation potential. The differentiation potential of ASCs is directed through manipulating culture medium composition with an introduction of growth factors to obtain the desired cell type. ASCs have been widely studied for its regenerative therapeutic solution to neurologic, skin, wound, muscle, bone, and other disorders. These therapeutic outcomes of ASCs are achieved possibly via autocrine and paracrine effects of their secretome comprising of cytokines, extracellular proteins and RNAs. Therefore, secretome-derivatives might offer huge advantages over cells through their synthesis and storage for long-term use. When considering the therapeutic significance and future prospects of ASCs, this review summarizes the recent developments made in harvesting, isolation, and characterization. Furthermore, this article also provides a deeper insight into secretome of ASCs mediating regenerative efficacy.

A simple strategy for retargeting lentiviral vectors to desired cell types via a disulfide-bond-forming protein-peptide pair

Despite recent improvements in the engineering of viral envelope proteins, it remains a significant challenge to create lentiviral vectors that allow targeted transduction to specific cell populations of interest. In this study, we developed a simple ‘plug and play’ strategy to retarget lentiviral vectors to any desired cell types through in vitro covalent modification of the virions with specific cell-targeting proteins (CTPs). This strategy exploits a disulfide bond-forming protein-peptide pair PDZ1 and its pentapeptide ligand (ThrGluPheCysAla, TEFCA). PDZ1 was incorporated into an engineered Sindbis virus envelope protein (Sind-PDZ1) and displayed on lentiviral particles while the TEFCA pentapeptide ligand was genetically linked to the CTP. Her2/neu-binding designed ankyrin repeat proteins (DARPin) were used as our model CTPs. DARPin-functionalized unconcentrated lentiviral vectors harboring Sind-PDZ1 envelope protein (Sind-PDZ1-pp) exhibited >800-fold higher infectious titer in HER2+ cells than the unfunctionalized virions (8.5 × 106 vs. <104 IU/mL). Moreover, by virtue of the covalent disulfide bond interaction between PDZ1 and TEFCA, the association of the CTP with the virions is nonreversible under non-reducing conditions (e.g. serum), making these functionalized virions potentially stable in an in vivo setting.

Chemical compound-based direct reprogramming for future clinical applications

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy.

Depletion of Dead Cells from Primary Tissue in 6 Minutes

Abstract Tissue specific research often requires mechanical and/or enzymatic digestion to isolate and study certain cell types. The digestion can be a harsh process resulting in a significant number of dead cells in the final cell suspension. Subsequent analysis by flow cytometry is difficult to interpret due to non-specific binding of antibodies to dead cells and dead cell auto fluorescence. Factors released by dead cells can also interfere with downstream assays, complicating the study of primary tissues. During apoptosis, the cell membrane loses its phospholipid asymmetry resulting in exposure of negatively charged phospholipids on the cell surface. Relocation of phosphatidylserine (PS) to the outer leaflet of the cell membrane is a well-established marker of apoptosis. By targeting exposed PS with Annexin V we have developed a rapid method (EasySep™) to immunomagnetically remove dead cells from primary tissue samples. Performance of this kit was examined on various mouse and human tissue types. Using this method, we were able to improve viability of a single cell suspension of mouse lungs digested with collagenase/hyaluronidase from an initial viability of 39.6 ± 12.3% AnxV−/PI− to 70.9 ± 11.8% AnxV−/PI. From 1×10^8 total start cells, 1.43 ± 0.68 ×10^7 live cells were recovered (n=10). From human polymorphonuclear leukocytes cultured overnight, viability was improved from 23.7 ± 9.8% AnxV−/PI− to 67.7 ± 12.1% AnxV−/PI− with recovery of 1.27 ± 0.52 ×10^7 live cells from 1×10^8 total start cells (n=7). This equates to removal of 88.9 ± 8.3% and 91.8 ± 5.3% dead cells respectively. Since live cells are untouched, subsequent isolation of desired cell types can be performed, resulting in a more viable population of cells for downstream applications.

Neural Stem Cell Differentiation Using Microfluidic Device-Generated Growth Factor Gradient

Neural stem cells (NSCs) have the ability to self-renew and differentiate into multiple nervous system cell types. During embryonic development, the concentrations of soluble biological molecules have a critical role in controlling cell proliferation, migration, differentiation and apoptosis. In an effort to find optimal culture conditions for the generation of desired cell types in vitro, we used a microfluidic chip-generated growth factor gradient system. In the current study, NSCs in the microfluidic device remained healthy during the entire period of cell culture, and proliferated and differentiated in response to the concentration gradient of growth factors (epithermal growth factor and basic fibroblast growth factor). We also showed that overexpression of ASCL1 in NSCs increased neuronal differentiation depending on the concentration gradient of growth factors generated in the microfluidic gradient chip. The microfluidic system allowed us to study concentration-dependent effects of growth factors within a single device, while a traditional system requires multiple independent cultures using fixed growth factor concentrations. Our study suggests that the microfluidic gradient-generating chip is a powerful tool for determining the optimal culture conditions.