Cationic Vectors Research Articles

Evolving Differentiated Local Polar Displacement and Relaxor Behavior in Pb(Mg1/3Nb2/3)O3–PbTiO3 Perovskites

Lead-based relaxor ferroelectrics feature exceptional dielectric and electromechanical properties. The inherent local structural inhomogeneity remains poorly characterized and impedes the understanding of their unique electrical behavior. Herein, the local structure of a technologically important 0.75Pb(Mg1/3Nb2/3)O3–0.25PbTiO3 relaxor is studied as a function of temperature, by employing neutron total scattering combined with newly advanced reverse Monte Carlo modeling. The statistical analysis of local cationic polar displacement vectors demonstrates that a prevailing order/disorder feature and strong displacive behavior are observed in Pb and Ti, but both weak components are observed in Nb/Mg. This leads to trifurcated polar behavior across the feature temperatures. Importantly, these distinct cationic polar vectors are correlated and present a microscopic picture, where sporadic correlated orthorhombic polar clusters are embedded in a long-range rhombohedral polar matrix at low temperature. Their fraction extends with increasing temperature, presenting strong ferroelectricity with dielectric relaxation macroscopically. Furthermore, the long-range rhombohedral polar ordering is disrupted by temperature, forming free-oriented polar clusters with reducing electric dipole length, gradually losing their macroscopic polarization, and resulting in a diffuse phase transition behavior. Altogether, this offers a fundamental basis in terms of local polar fluctuations in inferring the nature of the unique properties of relaxor ferroelectrics.

Controlled preparation of cholesterol-bearing polycations with pendent l-lysine for efficient gene delivery

A new series of cholesterol-bearing poly(methacrylate)s with pendant cationic (l-)-lysine (Chol-PHML) was synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization, followed by Boc-deprotection. Their structures were characterized by nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). Then, the average particle sizes and zeta potentials of the Chol-PHML/pDNA polyplexes were examined using dynamic light scattering (DLS), and their plasmid DNA (pDNA)-binding ability was analyzed using agarose gel retardation assay. In vitro cytotoxicity of the polycations was determined using a cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) kits in HeLa cells. The polyplex’s gene transfection effectiveness was investigated using a luciferase assay. The results revealed that the Chol-PHML series could efficiently bind/load pDNA with a high binding affinity (N/P < 3.0) and subsequently form nanoparticles with the sizes of 90–200 nm and zeta potentials of ∼+40 mV in an aqueous solution. The in vitro cytotoxicity and gene transfection efficiency considerably depended on the Chol-PHML vector’s molecular weight. The optimal Chol-PHML27 exhibited a substantially higher gene transfection efficiency and lower cytotoxicity than those of the linear structural PLL (15 K–30K) and PHML26 control. This study presents a simple method for synthesizing biocompatible and controllable cationic gene vectors by introducing hydrophobic cholesterol moieties, which will function as a promising foundation for gene delivery.

Small Molecule Modifications Significantly Increase the Transfection Efficiency of Low-Molecular Polymer.

Gene vectors with high biocompatibility and transfection efficiency are critical for successful gene therapy. PEI 25K (Polyethyleneimine 25K) is a common polymeric gene vector that has been employed as a positive control material in gene transfection studies due to its good performance in endosome escape. PEI 25K's indegradability and abundance of positive charges, on the other hand, cause toxicity in cells, limiting its use. In this study, we developed the PEI-ER non-viral vector by adding an endoplasmic reticulum (ER) targeting ligand to low molecular weight PEI 1.8K. These small molecule modifications dramatically improved PEI transfection efficiency while barely interfering with compatibility. PEI-ER/DNA complexes were discovered to enter the cell via caveolin-mediated endocytosis, avoiding destruction in the endosome. We believe that this little chemical alteration is a simple solution to enhance the efficacy of cationic polymer vectors in gene transport, and it has a lot of medicinal applications.

Nicotinic acid-based cationic vectors for efficient gene delivery to glioblastoma cells

A tocopherol-conjugated nicotinic acid-based lipid (NGT) was used for liposomal formation with the co-lipid DOPE and exhibited enhanced transfection of glioblastoma cells for eGFP and β-galactosidase protein expression.

Competitive binding and molecular crowding regulate the cytoplasmic interactome of non-viral polymeric gene delivery vectors

In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

Controlled DNA Delivery Using Poly(lactide) Nanoparticles and Understanding the Binding Interactions.

Cationic polymer-based gene delivery vectors suffer from several limitations such as low DNA-loading capacity, poor transfection, toxicity, environmental degradations, etc. Again, very limited works are available addressing the binding interactions in detail at the atomic level explaining the loading capacity, protection ability against harsh environments, and controlled release behavior of the DNA-encapsulated vehicles. Here, a poly(l-lactide) (PLA) nanoparticle-based controlled DNA release system is proposed. The developed vehicle possesses a high DNA-loading capacity and can release the loaded DNA in a controlled manner. Spectroscopic, physicochemical, and molecular simulation techniques (AM1 and atomistic molecular dynamics) have been employed to understand the binding interactions between PLA and DNA molecules enabling high DNA loading, protection against external harsh environments, and controlled DNA release behavior. Methyl thiazolyl tetrazolium (MTT) assay experiments confirm the biocompatible nature of the vehicle. Cellular uptake efficiency and endo-lysosomal escape capabilities have been investigated against HeLA cells. This study, therefore, demonstrates the development of a promising nonviral DNA delivery vector and includes a detailed investigation of the atomic-level interaction behavior between PLA and DNA molecules.

A Fluorescent Self-Reporting Vector with GSH Reduction Responsiveness for Nucleic Acid Delivery.

Nonviral gene vectors with stimulus responsiveness and self-reporting properties have broad application prospects in gene therapy. Herein, we developed a nanosized reduction-responsive cationic liposomal vector formed from DNS(Zn), in which a naphthalimide-sulfonamide group was used as the glutathione (GSH)-responsive group to generate a blue fluorescence signal at 458 nm. Macrocyclic polyamine (cyclen) was used as a cationic headgroup to facilitate Zn(II) coordination, which may reduce the cytotoxicity and improve transfection efficiency. The Zn-free and nonresponsive analogues were used for comparison. Fluorescent assays revealed that the GSH response of DNS(Zn) could increase the blue fluorescence signal and improve the DNA release in cells. The title material also showed higher positive ζ-potential than its nonresponsive analogue, resulting in stronger DNA binding ability and better cellular uptake. These advantages made DNS(Zn) a good candidate for nonviral gene delivery, and the transfection efficiency in HeLa cells was distinctly higher than that of its analogue and the commercially available transfection reagent. Besides plasmid DNA, DNS(Zn) could also deliver small interfering RNA (siRNA) with good gene silencing efficiency, extending the application of the liposomes. These results suggest that DNS(Zn) can serve as a highly efficient nucleic acid delivery vector with reduction-responsive fluorescence self-reporting ability in tumor cells.

Paclitaxel loading in cationic liposome vectors is enhanced by replacement of oleoyl with linoleoyl tails with distinct lipid shapes

Lipid carriers of hydrophobic paclitaxel (PTX) are used in clinical trials for cancer chemotherapy. Improving their loading capacity requires enhanced PTX solubilization. We compared the time-dependence of PTX membrane solubility as a function of PTX content in cationic liposomes (CLs) with lipid tails containing one (oleoyl; DOPC/DOTAP) or two (linoleoyl; DLinPC/newly synthesized DLinTAP) cis double bonds by using microscopy to generate kinetic phase diagrams. The DLin lipids displayed significantly increased PTX membrane solubility over DO lipids. Remarkably, 8 mol% PTX in DLinTAP/DLinPC CLs remained soluble for approximately as long as 3 mol% PTX (the solubility limit, which has been the focus of most previous studies and clinical trials) in DOTAP/DOPC CLs. The increase in solubility is likely caused by enhanced molecular affinity between lipid tails and PTX, rather than by the transition in membrane structure from bilayers to inverse cylindrical micelles observed with small-angle X-ray scattering. Importantly, the efficacy of PTX-loaded CLs against prostate cancer cells (their IC50 of PTX cytotoxicity) was unaffected by changing the lipid tails, and toxicity of the CL carrier was negligible. Moreover, efficacy was approximately doubled against melanoma cells for PTX-loaded DLinTAP/DLinPC over DOTAP/DOPC CLs. Our findings demonstrate the potential of chemical modifications of the lipid tails to increase the PTX membrane loading while maintaining (and in some cases even increasing) the efficacy of CLs. The increased PTX solubility will aid the development of liposomal PTX carriers that require significantly less lipid to deliver a given amount of PTX, reducing side effects and costs.

Dopamine-Grafted Hyaluronic Acid Coated Hyperbranched Poly(β-Amino Esters)/DNA Nano-Complexes for Enhanced Gene Delivery and Biosafety

Gene therapy has attracted particular attention for the treatment of various genetic diseases, and the development of gene delivery vectors is of utmost importance for in vivo applications of gene drugs. Various cationic polymers with high nucleic acid loading and intracellular transfection efficiency have been reported, however, their biological applications are limited by potential toxicity. Surface modification is a robust solution to detoxify the cationic vectors, but this can inevitably weaken the transfection efficiency. To address this dilemma, we reported the ability of a dopamine (DA)-grafted hyaluronic acid (HA) to modify gene vectors for enhanced gene delivery and biosafety. The nano-vector was formed by using branched poly(β-amino esters) (PAEs), and surface coating with HA-DA to form a core-shell nano-structure via electrostatic attraction. Upon HA-DA modification, the biosafety of the gene delivery vehicle was improved, as demonstrated by the cell cytotoxicity assay and hemolysis test. Notably, the nano-system displayed a DA-dependent transfection efficiency, in which a higher DA grafting degree resulted in better efficacy. This can be explained by the adhesive nature of DA, facilitating cell membrane interaction, as well as DA receptor mediated active targeting. At the optimal DA grafting ratio, the nano-system achieved a transfection efficiency even better than that of commonly used polyethylenimine (PEI) vectors. Together with its excellent biocompatibility, the vector presented here holds great promise for gene delivery applications.

Preparation and Biological Property Evaluation of Novel Cationic Lipid-Based Liposomes for Efficient Gene Delivery.

Novel cationic lipid-based liposomes prepared using an amphiphilic cationic lipid material, N,N-dimethyl-(N',N'-di-stearoyl-1-ethyl)1,3-diaminopropane (DMSP), have been proposed to enhance the transfection of nucleic acids. Herein, we designed and investigated liposomes prepared using DMSP, soybean phosphatidylcholine, and cholesterol. This novel gene vector has high gene loading capabilities and excellent protection against nuclease degradation. An in vitro study showed that the liposomes had lower toxicity and superior cellular uptake and transfection efficiency compared with Lipofectamine 2000. An endosomal escape study revealed that the liposomes demonstrated high endosomal escape and released their genetic payload in the cytoplasm efficiently. Mechanistic studies indicated that the liposome/nucleic acid complexes entered cells through energy-dependent endocytosis that was mediated by fossa proteins. These results suggest that such cationic lipid-based liposome vectors have potential for clinical gene delivery.

Polyethylenimine-based nanovector grafted with mannitol moieties to achieve effective gene delivery and transfection

Polyethylenimine (PEI), a kind of cationic non-viral gene delivery vector, is capable of stable and efficient transgene expression for gene delivery. However, low transfection efficiency in vivo along with high toxicity limited the further application of gene therapy in the clinic. To enhance gene transfection performance and reduce cytotoxicity of polyethylenimine, branched polyethylenimine-derived cationic polymers BPEI25 k-man-S/L/M/H with different grafting degree with mannitol moieties were prepared and the transfection efficiency was evaluated. Among them, BPEI25 k-man-L showed the best transfection efficiency, lower toxicity, and significantly enhanced long-term systemic transgene expression for 96 h in vivo even at a single-dose administration. The results of cellular uptake mechanism and western-blot experiments revealed that the mannitol modification of BPEI25 k induced and up-regulated the phosphorylation of caveolin-1 and thus enhanced the caveolae-mediated cellular uptake. This class of gene delivery system highlights a paradigmatic approach for the development of novel and safe non-viral vectors for gene therapy.

Cationic Polymer Nanoparticles-Mediated Delivery of miR-124 Impairs Tumorigenicity of Prostate Cancer Cells.

MicroRNAs (miRNAs) play a pivotal role in regulating the expression of genes involved in tumor development, invasion, and metastasis. In particular, microRNA-124 (miR-124) modulates the expression of carnitine palmitoyltransferase 1A (CPT1A) at the post-transcriptional level, impairing the ability of androgen-independent prostate cancer (PC3) cells to completely metabolize lipid substrates. However, the clinical translation of miRNAs requires the development of effective and safe delivery systems able to protect nucleic acids from degradation. Herein, biodegradable polyethyleneimine-functionalized polyhydroxybutyrate nanoparticles (PHB-PEI NPs) were prepared by aminolysis and used as cationic non-viral vectors to complex and deliver miR-124 in PC3 cells. Notably, the PHB-PEI NPs/miRNA complex effectively protected miR-124 from RNAse degradation, resulting in a 30% increase in delivery efficiency in PC3 cells compared to a commercial transfection agent (Lipofectamine RNAiMAX). Furthermore, the NPs-delivered miR-124 successfully impaired hallmarks of tumorigenicity, such as cell proliferation, motility, and colony formation, through CPT1A modulation. These results demonstrate that the use of PHB-PEI NPs represents a suitable and convenient strategy to develop novel nanomaterials with excellent biocompatibility and high transfection efficiency for cancer therapy.

Electrical Field-Assisted Gene Delivery from Polyelectrolyte Multilayers.

To sustain gene delivery and elongate transgene expression, plasmid DNA and cationic nonviral vectors can be deposited through layer-by-layer (LbL) assembly to form polyelectrolyte multilayers (PEMs). Although these macromolecules can be released for transfection purposes, their entanglement only allows partial delivery. Therefore, how to efficiently deliver immobilized genes from PEMs remains a challenge. In this study, we attempt to facilitate their delivery through the pretreatment of the external electrical field. Multilayers of polyethylenimine (PEI) and DNA were deposited onto conductive polypyrrole (PPy), which were placed in an aqueous environment to examine their release after electric field pretreatment. Only the electric field perpendicular to the substrate with constant voltage efficiently promoted the release of PEI and DNA from PEMs, and the higher potential resulted in the more releases which were enhanced with treatment time. The roughness of PEMs also increased after electric field treatment because the electrical field not only caused electrophoresis of polyelectrolytes and but also allowed electrochemical reaction on the PPy electrode. Finally, the released DNA and PEI were used for transfection. Polyplexes were successfully formed after electric field treatment, and the transfection efficiency was also improved, suggesting that this electric field pretreatment effectively assists gene delivery from PEMs and should be beneficial to regenerative medicine application.

Regulating intracellular fate of siRNA by endoplasmic reticulum membrane-decorated hybrid nanoplexes

Most cationic vectors are difficult to avoid the fate of small interfering RNA (siRNA) degradation following the endosome-lysosome pathway during siRNA transfection. In this study, the endoplasmic reticulum (ER) membrane isolated from cancer cells was used to fabricate an integrative hybrid nanoplexes (EhCv/siRNA NPs) for improving siRNA transfection. Compared to the undecorated Cv/siEGFR NPs, the ER membrane-decorated EhCv/siRNA NPs exhibits a significantly higher gene silencing effect of siRNA in vitro and a better antitumor activity in nude mice bearing MCF-7 human breast tumor in vivo. Further mechanistic studies demonstrate that functional proteins on the ER membrane plays important roles on improving cellular uptake and altering intracellular trafficking pathway of siRNA. It is worth to believe that the ER membrane decoration on nanoplexes can effectively transport siRNA through the endosome-Golgi-ER pathway to evade lysosomal degradation and enhance the silencing effects of siRNA.

Interparticle Interactions in Dilute Solutions of Polyelectrolyte Complex Micelles.

The application of dilute solutions of polyelectrolyte complex (PEC) micelles for delivering therapeutic nucleic acids into disease sites has gained momentum. This Letter reports a detailed characterization of PEC micelles in dilute solutions including their internal structures and the determination of the interparticle interactions. The polymer concentration ranges from 0.1 to 0.5 wt %, a regime where micelle-micelle interactions are infrequent. We employ synchrotron small-angle X-ray scattering (SAXS) to simultaneously probe the morphology, internal structure, and radius of gyration (Rg) of the self-assemblies formed by charged diblock polyelectrolytes and homopolyelectrolytes. The emerging appearance of the structure factor in SAXS profiles with the increasing polymer concentration demonstrates the presence of the repulsive intermicellar correlations, which is further confirmed by the differences between the "reciprocal Rg" estimated by Guinier approximation and the "real space Rg" determined by pair distribution functions. We find that the soft corona chains tethered on the surface of phase-separated complex domains are compressed when micelles come closeto thepointwhere a hard-sphere interaction takes over. These findings contribute to the fundamental understanding of the structure and space-filling constraints in the complexation-driven self-assemblies and advance the rational design of cationic polymer-based nonviral gene delivery vectors.

Halloysite nanotubes-carbon dots hybrids multifunctional nanocarrier with positive cell target ability as a potential non-viral vector for oral gene therapy

HypothesisThe use of non-viral vectors for gene therapy is hindered by their lower transfection efficiency and their lacking of self-track ability. ExperimentsThis study aims to investigate the biological properties of halloysite nanotubes-carbon dots hybrid and its potential use as non-viral vector for oral gene therapy. The morphology and the chemical composition of the halloysite hybrid were investigated by means of high angle annular dark field scanning TEM and electron energy loss spectroscopy techniques, respectively. The cytotoxicity and the antioxidant activity were investigated by standard methods (MTS, DPPH and H2O2, respectively) using human cervical cancer HeLa cells as model. Studies of cellular uptake were carried out by fluorescence microscopy. Finally, we investigated the loading and release ability of the hybrid versus calf thymus DNA by fluorescence microscopy, circular dichroism, dynamic light scattering and ζ-potential measurements. FindingsAll investigations performed confirmed the existence of strong electrostatic interactions between the DNA and the halloysite hybrid, so it shows promise as a multi-functional cationic non-viral vector that has also possesses intracellular tracking capability and promising in vitro antioxidant potential.

Nuclear delivery of plasmid DNA determines the efficiency of gene expression.

As a cationic non-viral gene delivery vector, poly(agmatine/ N, N'-cystamine-bis-acrylamide) (AGM-CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM-CBA/pDNA polyplexes was found to have a non-linear relationship with AGM-CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM-CBA), we used pGL3-control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM-CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate-limiting step for pLUC transfection expression. Further optimization of the non-viral gene delivery system can be focused on the improvement of gene intracellular availability.

Dex-Aco coating simultaneously increase the biocompatibility and transfection efficiency of cationic polymeric gene vectors

Cationic polymeric vectors attracted plenty of attentions in gene therapy due to nonimmunogenicity, easy to synthesis and flexible properties. However, biocompatibility challenge such as nonspecific interactions with blood cells and serum proteins, may affect the delivery efficiency of cationic vectors; besides, inefficient endosomal escape causes low transfection efficiency. Herein, we synthesized an anionic coating polymer dextran-g-aconic anhydride (Dex-Aco, DA) through a simple esterification reaction, which can protect cationic polymer poly(cystamine-bis-acrylamide)-agmatine-histamine (PCAH, PC) constructed nanomedicine against interactions with blood cells and serum proteins, improving biocompatibility. Interestingly, DA coating significantly increased the transfection efficiency of cationic PC，not due to the increase of cellular uptake, nor functioning as a receptor ligand, but was associated to the change of endocytosis pathway. Finally, using programmed cell death protein 4 (PDCD4) as a functional gene, DA coating PC NPs showed improved therapeutic effect and biocompatibility on tumor bearing mice. We believe that this DA coating PC NPs provides a facile method to improve the performance of cationic polymer vectors in gene therapy and has great potential for clinical applications.

A Kinetic Model of Oligonucleotide-Brush Interactions for the Rational Design of Gene Delivery Vectors.

Polymer brushes are attractive candidates for the design of gene delivery vectors as they allow the systematic study of the impact of structural (type, size, and shape of nanomaterials core) and physicochemical parameters (cationic monomer chemistry, brush thickness, and grafting density) on transfection efficiency. However, relatively little is known of their interactions of oligonucleotides. To study such interactions, we use surface plasmon resonance and developed a kinetic model of brush binding and infiltration. We identify the striking impact that brush grafting density and thickness have on oligonucleotide kinetics of infiltration, binding affinity, and maximum loading. Surprisingly, double-stranded RNA molecules are found to load at significantly higher levels compared to DNA molecules of identical sequence (apart from uracils/thymines). Furthermore, analysis of the kinetics of adsorption of these oligonucleotides indicates that the stoichiometry of binding (ratio of amine versus phosphate residues) is close to parity for the uptake of 20 bp double-stranded RNA. Finally, nanoparticles were designed to be used as gene transfection vectors and to quantify if the brush grafting density and thickness significantly impact transfection efficiencies in a small interfering RNA knockdown assay. Therefore, this study demonstrates the rational design of polymer brush-based nanoparticle vectors for efficient delivery of oligonucleotides. The model developed will allow to uncover how the refinement of the physicochemical and structural properties of polymer brushes enable the tuning of RNA binding and allow the systematic study of cationic vectors efficiency for RNA delivery.

The planarian Schmidtea mediterranea as a model system for the discovery and characterization of cell-penetrating peptides and bioportides.

The general utility of the planarian Schmidtea mediterranea, an organism with remarkable regenerative capacity, was investigated as a convenient three-dimensional model to analyse the import of cell-penetrating peptides (CPPs) and bioportides (bioactive CPPs) into complex tissues. The unpigmented planarian blastema, 3days post head amputation, is a robust platform to assess the penetration of red-fluorescent CPPs into epithelial cells and deeper tissues. Three planarian proteins, Ovo, ZicA and Djeya, which collectively control head remodelling and eye regeneration following decapitation, are a convenient source of novel cationic CPP vectors. One example, Djeya1 (RKLAFRYRRIKELYNSYR), is a particularly efficient and seemingly inert CPP vector that could be further developed to assist the delivery of bioactive payloads across the plasma membrane of eukaryotic cells. Eye regeneration, following head amputation, was utilized in an effort to identify bioportides capable of influencing stem cell-dependent morphogenesis. These investigations identified the tetradecapeptide mastoparan (INLKALAALAKKIL) as a bioportide able to influence the gross morphology of head development. We conclude that, compared with cellular monolayers, the S.mediterranea system provides many advantages and will support the identification of bioportides able to selectively modify the biology of totipotent neoblasts and, presumably, other mammalian stem cell types.