Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells

We investigated the localization and functional expression of the ABC transporter ABCB1 in human fetal neural stem/progenitor cells (hNSPCs). RT-PCR analysis revealed ABCB1 gene expression in hNSPCs. We found a single band in immunoblotted hNSPCs lysates probed with ABCB1 antibody, and detected ABCB1 at the hNSPCs cell membrane by immunocytochemistry and subcellular fractionation. ABCB1 inhibitors and substrate, and ATP-depleting agents enhanced hNSPCs' rhodamine 123 accumulation, and hNSPCs microsomes had vanadate-sensitive ATPase activity. ABCB1 and nestin expression decreased during hNSPCs differentiation, while the astroglial marker GFAP increased. ABCB1 may maintain hNSPCs in an undifferentiated state and could be a neural stem/progenitor marker.     

Transfecting human neural stem cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency. Other mechanical methods of introducing a gene of interest, such as a "gene gun" or microinjection, are also limited by poor cell viability and low numbers of transfected cells. The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns. We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells. Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection. The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.     

The potential of stem cells for the treatment of brain tumors and globoid cell leukodystrophy

Stem cells of different origin are under careful scrutiny as potential new tools for the treatment of several neurological diseases. The major focus of these reaserches have been neurodegenerative disorders, such as Huntington Chorea or Parkinson Disease (Shihabuddin et al., 1999). More recently attention has been devoted to their use for brain repair after stroke (Savitz et al., 2002). In this review we will focus on the potential of stem cell treatments for glioblastoma multiforme (Holland, 2000), the most aggressive primary brain tumor, and globoid cell leukodystrophy (Krabbe disease), a metabolic disorder of the white matter (Berger et al., 2001). These two diseases may offer a paradigm of what the stem cell approach may offer in term of treatment, alone or in combination with other therapeutic approaches. Two kinds of stem cells will be consideredhere: neural stem cells and hematopoietic stem cells, both obtained after birth. The review will focus on experimental models, with an eye on clinical perspectives. This revised version was published online in July 2006 with corrections to the Cover Date.     

Converted neural cells: induced to a cure?

Many neurodegenerative disorders such as Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) and others often occur as a result of progressive loss of structure or function of neurons. Recently, many groups were able to generate neural cells, either differentiated from induced pluripotent stem cells (iPSCs) or converted from somatic cells. Advances in converted neural cells have opened a new era to ease applications for modeling diseases and screening drugs. In addition, the converted neural cells also hold the promise for cell replacement therapy (Kikuchi et al., 2011; Krencik et al., 2011; Kriks et al., 2011; Nori et al., 2011; Rhee et al., 2011; Schwartz et al., 2012). Here we will mainly discuss most recent progress on using converted functional neural cells to treat neurological diseases and highlight potential clinical challenges and future perspectives.     

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Hypoxic culture and in vivo inflammatory environments affect the assumption of pericyte characteristics by human adipose and bone marrow progenitor cells

Previous studies have shown that exposure to a hypoxic in vitro environment increases the secretion of pro-angiogenic growth factors by human adipose-derived stromal cells (hASCs) [Cao Y, et al., Biochem Biophys Res Commun 332: 370-379, 2005; Kokai LE, et al., Plast Reconstr Surg 116: 1453-1460, 2005; Park BS, et al., Biomed Res (Tokyo) 31: 27-34, 2010; Rasmussen JG, et al., Cytotherapy 13: 318-328, 2010; Rehman J, et al., Circulation 109: 1292-1298, 2004]. Previously, it has been demonstrated that hASCs can differentiate into pericytes and promote microvascular stability and maintenance during angiogenesis in vivo (Amos PJ, et al., Stem Cells 26: 2682-2690, 2008; Traktuev DO, et al., Circ Res 102: 77-85, 2008). In this study, we tested the hypotheses that angiogenic induction can be increased and pericyte differentiation decreased by pretreatment of hASCs with hypoxic culture and that hASCs are similar to human bone marrow-derived stromal cells (hBMSCs) in these regards. Our data confirms previous studies showing that hASCs: 1) secrete pro-angiogenic proteins, which are upregulated following culture in hypoxia, and 2) migrate up gradients of PDGF-BB in vitro, while showing for the first time that a rat mesenteric model of angiogenesis induced by 48/80 increases the propensity of both hASCs and hBMSCs to assume perivascular phenotypes following injection. Moreover, culture of both cell types in hypoxia before injection results in a biphasic vascular length density response in this model of inflammation-induced angiogenesis. The effects of hypoxia and inflammation on the phenotype of adult progenitor cells impacts both the therapeutic and the basic science applications of the cell types, as hypoxia and inflammation are common features of natural and pathological vascular compartments in vivo.     

Suspended in culture--human pluripotent cells for scalable technologies

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively termed human pluripotent stem cells (hPSCs), are typically derived and maintained in adherent and semi-defined culture conditions. Recently a number of groups, including Chen et al., 2012, have demonstrated that hESCs can now be expanded efficiently and maintain pluripotency over long-term passaging as aggregates in a serum-free defined suspension culture system, permitting the preparation of scalable cGMP derived hPSC cultures for cell banking, high throughput research programs and clinical applications. In this short commentary we describe the utility and potential future uses of suspension culture systems for hPSCs.     

Development of a novel beta-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures

Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.     

Non-immortalized human neural stem (NS) cells as a scalable platform for cellular assays

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells ( [Conti et al., 2005] and [Sun et al., 2008]). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133⁺CD24^−/lo cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca²⁺ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny.     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

Influence of chromatographic fractions of extracts derived from bovine neural retina on newt (Notophthalmus viridescens) lens regeneration in vitro

Removal of the ocular lens in adult newts (Notophthalmus viridescens) is followed by a series of cellular events leading to regeneration of a new lens by cell type conversion of pigmented iris epithelial cells at the dorsal pupillary margin (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). Following depigmentation and five to seven cell divisions, iris epithelial cells redifferentiate into lens fiber cells and synthesize crystallin proteins (Yamada, Curr. Top. Dev. Biol. 2:247-283, 1967). This process is dependent upon neural retina in vivo (Stone, Anat. Rec. 131:151-172, 1958; Reyer, Dev. Biol. 14:214-225, 1966) and in vitro (Yamada et al., Differentiation 1:65-82, 1973). Acting on the hypothesis that the role of the neural retina is to promote passage of iris epithelial cells through the requisite number of cell cycles which will then allow them to redifferentiate as lens fiber cells (Yamada, in: Cell Biology of the Eye. Academic Press, New York, 1982), we undertook testing of the effects of eye-derived mitogenic substances, as well as other mitogens, on regeneration of lens from iris in organ culture. We have previously defined a critical period for the retinal influence in vivo and in vitro, and have shown that crude extracts of retina can enhance regeneration of lenses in culture (Connelly et al., J. Exp. Zool., 240:343-351, 1986). In this paper, we report on the lens regeneration enhancing activity (LRA) of more highly purified fractions of the retinal extracts. Heparin-sepharose chromatography of the crude retinal extract yields three fractions (Courty et al., Biochemie 67:265-269, 1985) called EDGF I, II, and III. EDGF I and II have affinity for heparin, while EDGF III does not. In our bioassay, LRA appears only in the EDGF III fraction. Dialysis of EDGF III against 0.1 N acetic acid yields a fraction which has affinity for cibacron blue sepharose (eluting at 2.15 M salt) and also has significant LRA. Because insulin at high doses has a marginal effect on lens regeneration in culture (Williams and McGlinn, Am. Zool. 19:923, 1979; Connelly, Differentiation 16:85-91, 1980), we tested IGF-I. Because of the putative neurotrophic effects of transferrin (Tf) (Mescher and Munaim, J. Exp. Zool., 230:485-490, 1986), we tested Tf for its ability to enhance regeneration of the lens in culture. IGF-I seems to have an enhancing effect on lens regeneration; Tf does not.     

Manipulating midbrain stem cell self-renewal

In this issue of Cell Stem Cell, Falk and colleagues (Falk et al., 2008) demonstrate that differential responsiveness to TGF-beta signaling selectively modulates self-renewal of dorsal midbrain stem cells. This observation may lead to strategies for expanding specific neural stem cell subtypes.     

Single-cell RNA-Seq analysis identified kidney progenitor cells from human urine

Dear Editor, Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra.Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases.Among them, renal tubular cells and podocytes have been identified and 2D or 3D cultured from human urine specimens (Oliveira Arcolino et al., 2015;Schutgens et al.,2019).Particularly, kidney stem cell/progenitor cells were successfully recovered from pediatric patient urine and then cultured for kidney regenerative purpose by the Romagnani group.However, they also showed that such cells cannot be recovered from healthy individuals (Lazzeri et al., 2015).It remains unknown whether similar types of progenitor cells can be found in different individuals, either healthy or diseased.     

Mobilized hematopoietic stem cell yield depends on species-specific circadian timing

Endogenous rhythmicity likely evolved as a mechanism allowing organisms to anticipate predictable daily changes in the environment (Rutter et al., 2002). Under homeostasis, murine hematopoietic stem cell (HSC) egress is orchestrated by rhythmic beta 3 adrenergic signals delivered by the sympathetic nervous system (SNS) that regulate Cxcl12 expression in stromal cells (Mendez-Ferrer et al., 2008). Here, we show that CXCR4 is also regulated under circadian control whose rhythm is synchronized with its ligand, CXCL12, to optimize HSC trafficking. These circadian oscillations are inverted in humans compared to the mouse and continue to influence the yield even when stem cell mobilization is enforced. Our results suggest that the human HSC yield for clinical transplantation might be significantly greater if patients were harvested during the evening compared to the morning.     

Derivation of Germline Competent Rat Embryonic Stem Cells from DA Rats

The laboratory rat was one of the earliest mammalian species for scientific research and used as animal disease models in physiology,toxicology,behavior,immunology,and tumor-biology for over 150 years (Jacob,1999).However,rat lags far behind mouse in generating human disease models and functional genomic studies because of the lack of authentic rat embryonic stem (ES) cells (Voigt and Serikawa,2009),whereas the first mouse ES cell line was established in 1981 (Evans and Kaufman,1981).By combining two or three kinase inhibitors which target GSK3,MEK and FGF signaling pathways in serum-free N2B27 medium,germline competent rat ES cells were first derived in 2008 (Buehr et al.,2008;Li et al.,2008).     

Food for thought: neural stem cells on a diet

Growing evidence shows that stem cells are modulated by systemic factors that are integrated with local signals in response to physiological status. Two recent Cell (Chell and Brand, 2010) and Nature (Sousa-Nunes et?al., 2011) papers reveal that Drosophila neural stem cell proliferation is controlled by a diet-dependent insulin/TOR signaling relay between tissues.     

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.