Peripheral Nerve Tissue Engineering Research Articles

A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model.

Over the past decade, silk fibroin (SF) has been emergently used in peripheral nerve tissue engineering. Current approaches aiming at producing SF-based nerve guidance conduits (SF-NGCs) used dissolved silk based on either aqueous solutions or organic solvents. In this study, we describe a novel procedure to produce SF-NGCs: A braided tubular structure of raw Bombyx mori silk is subsequently processed with the ternary solvent CaCl2/H2O/ethanol, formic acid, and methanol to improve its mechanical and topographical characteristics. Topographically, the combination of the treatments results in a fusion of the outer single silk fibers to a closed layer with a thickness ranging from about 40 to 75 μm. In contrast to the outer wall, the inner lumen (not treated with processing solvents) still represents the braided structure of single fibers. Mechanical stability, elasticity, and kink characteristics were evaluated with a custom-made test system. The modification procedure described here drastically improved the elastic properties of our tubular raw scaffold, favoring its use as a NGC. A cell migration assay with NIH/3T3-fibroblasts revealed the impermeability of the SF-NGC wall for possible invading and scar-forming cells. Moreover, the potential of the SF-NGC to serve as a substratum for Schwann cells has been demonstrated by cytotoxicity tests and live-dead stainings of Schwann cells grown on the inner surface of the SF-NGC. In vivo, the SF-NGC was tested in a rat sciatic nerve injury model. In short-term in vivo studies, it was proved that SF-NGCs are not triggering host inflammatory reactions. After 12 weeks, we could demonstrate morphological and functional reinnervation of the distal targets. Filled with collagen, a higher number of axons could be found in the distal to the graft (1678±303), compared with the empty SF-NGC (1274±146). The novel SF-NGC presented here shows promising results for the treatment of peripheral nerve injuries. The modification of braided structures to adapt their mechanical and topographical characteristics may support the translation of SF-based scaffolds into the clinical setting. However, further improvements and the use of extracellular matrix molecules and Schwann cells are suggested to enable silk tube based conduits to bridge long-distance nerve gaps.

Bioplotting Alginate/Hyaluronic Acid Hydrogel Scaffolds with Structural Integrity and Preserved Schwann Cell Viability

Bioplotting is an emerging freeform scaffold fabrication technique useful for creating artificial tissue scaffolds containing living cells. Simultaneous maintenance of scaffold structural integrity and cell viability is a challenging task. In this article, we present strategies developed to bioplot alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells for potential use in peripheral nerve tissue engineering. The fabrication platform, upon which the scaffold is created, was coated with the polycation polyethylenimine to immobilize the first layer of the scaffold on the platform. Each layer was then dispensed into a bath containing calcium chloride to cross-link the alginate, polyvinyl alcohol, and low concentrations of polyethylenimine to improve the structural integrity of the resulting scaffolds while retaining biocompatibility. The present study illustrated that with these strategies, porous alginate/hyaluronic acid scaffolds can be fabricated with good structural integrity and long-term cell viability. Plotting method and speed were also important factors determining the printability of single-layered scaffolds. The methods developed and the results obtained from this study form the basis for a general bioplotting method by which more sophisticated alginate/hyaluronic acid scaffolds can be fabricated with the inclusion of living cells into desired structures.

Gelatin-based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering.

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

In VitroEvaluation of Cell-Seeded Chitosan Films for Peripheral Nerve Tissue Engineering

Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)--immortalized, neonatal, and adult-of the chitosan substrate. Both cell types were viable on the biomaterial and showed different metabolic activities and proliferation behavior, indicating cell-type-specific cell-biomaterial interaction. Moreover, the cell types also displayed their typical morphology. In cocultures adult SCs used the BMSCs as a feeder layer and no negative interactions between both cell types were detected. Further, the chitosan films allow neurite outgrowth from dissociated sensory neurons, which is additionally supported on film preseeded with SC-BMSC cocultures. The presented chitosan films therefore demonstrate high potential for their use in tissue-engineered nerve grafts.

Development and characterization of novel agar and gelatin injectable hydrogel as filler for peripheral nerve guidance channels

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear-thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin-based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar-gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear-thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two- and three-dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

Comparison of morphology and biocompatibility of acellular nerve scaffolds processed by different chemical methods

To investigate the morphological differences among acellular rat nerve scaffolds processed by different chemical methods and compare the biocompatibility between rat nerve grafts processed by different chemical methods and rat adipose-derived stem cells in vitro. Acellular rat sciatic nerve scaffolds processed by two different chemical methods (the Sondell method and the optimized method) and normal rat sciatic nerves were used as control. The structure and components of nerve scaffold were observed under microscopy, the degrees of decellularization and demyelination of nerve scaffold and integrity of nerve fiber tubes were assessed. The rat adipose-derived stem cells growth and adherence on scaffold were studied by scanning electron microscopy, the activity and adhesive ratio of rat adipose-derived stem cells in the nerve scaffold were compared. The basal lamina tubes and the extracellular matrix in the epineurium and perineurium in the nerve graft of optimized method were better preserved than the nerve graft of the Sondell method. After co-cultured with scaffolds, the difference of cell activity between three groups (two cell-scaffold combinations and control group) at the same observation time were not statistically significant (P > 0.05),the adhesive ratio of rat adipose-derived stem cells in the scaffold of the optimized method was better than that of the Sondell method. The scaffold of the optimized method is more effective than the scaffold of the Sondell method for peripheral nerve tissue engineering.

Dorsal root ganglion-derived Schwann cells combined with poly(lactic-co-glycolic acid)/chitosan conduits for the repair of sciatic nerve defects in rats.

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and purification of Schwann cells are complicated by contamination with fibroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly purified Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells (> 95%) using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly(lactic-co-glycolic acid)/chitosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were significantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental findings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly(lactic-co-glycolic acid)/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.

Ultrasound imaging of chitosan nerve conduits that bridge sciatic nerve defects in rats.

Ultrasound imaging of chitosan nerve conduits that bridge sciatic nerve defects in rats.

Histological assessment in peripheral nerve tissue engineering.

The histological analysis of peripheral nerve regeneration is one of the most used methods to demonstrate the success of the regeneration through nerve conduits. Nowadays, it is possible to evaluate different parameters of nerve regeneration by using histological, histochemical, immunohistochemical and ultrastructural techniques. The histochemical methods are very sensible and are useful tools to evaluate the extracellular matrix remodeling and the myelin sheath, but they are poorly specific. In contrast, the immunohistochemical methods are highly specific and are frequently used for the identification of the regenerated axons, Schwann cells and proteins associated to nerve regeneration or neural linage. The ultrastructural techniques offer the possibility to perform a high resolution morphological and quantitative analysis of the nerve regeneration. However, the use of a single histological method may not be enough to assess the degree of regeneration, and the combination of different histological techniques could be necessary.

Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve.

Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, but cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhesion stage. Moreover, seeded cells were distributed throughout the material.

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells-derived neural crest stem cells (iPSCs-NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs-NCSCs were examined respectively in this study. LIPUS at 500 mW cm(-2) enhanced the viability and proliferation of iPSCs-NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs-NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs-NCSCs in vitro for peripheral nerve tissue engineering.

Repairing nerve gaps by vein conduits filled with lipoaspirate-derived entire adipose tissue hinders nerve regeneration

In spite of great recent advancements, the definition of the optimal strategy for bridging a nerve defect, especially across long gaps, still remains an open issue since the amount of autologous nerve graft material is limited while the outcome after alternative tubulization techniques is often unsatisfactory. The aim of this study was to investigate a new tubulization technique based on the employment of vein conduits filled with whole subcutaneous adipose tissue obtained by lipoaspiration. In adult rats, a 1cm-long defect of the left median nerve was repaired by adipose tissue-vein-combined conduits and compared with fresh skeletal muscle tissue-vein-combined conduits and autologous nerve grafts made by the excised nerve segment rotated by 180°. Throughout the postoperative period, functional recovery was assessed using the grasping test. Regenerated nerve samples were withdrawn at postoperative month-6 and processed for light and electron microscopy and stereology of regenerated nerve fibers. Results showed that functional recovery was significantly slower in the adipose tissue-enriched group in comparison to both control groups. Light and electron microscopy showed that a large amount of adipose tissue was still present inside the vein conduits at postoperative month-6. Stereology showed that all quantitative morphological predictors analyzed performed significantly worse in the adipose tissue-enriched group in comparison to the two control groups. On the basis of this experimental study in the rat, the use of whole adipose tissue for tissue engineering of peripheral nerves should be discouraged. Pre-treatment of adipose tissue aimed at isolating stromal vascular fraction and/or adipose derived stem/precursor cells should be considered a fundamental requisite for nerve repair.

Strategic Design and Recent Fabrication Techniques for Bioengineered Tissue Scaffolds to Improve Peripheral Nerve Regeneration

Bioengineered tissue scaffolds are a potential tool for improving regenerative repair of damaged peripheral nerves. Novel modes of fabrication coupled with scaffold design strategies that are based on an understanding of the biology of nerve injury offer the prospect of intervention at a more sophisticated level. We review the etiology and incidence of peripheral nerve injury and the biological events that unfold during nerve regeneration after an injury. Newly available tissue scaffold fabrication technologies using bioplotting and laser-based techniques are described. Scaffold design strategies are also discussed, including the incorporation of living cells during scaffold fabrication, inclusion of neurotrophic gradients, use of electric stimulation, inclusion of antioxidant compounds to counteract neural apotosis, and promotion of angiogenesis. Use of these advanced fabrication techniques and incorporation of one or more of these active biological strategies may eventually lead to a greater success in peripheral nerve tissue engineering.

Electrospun bio‐composite P(LLA‐CL)/collagen I/collagen III scaffolds for nerve tissue engineering

One of the biggest challenges in peripheral nerve tissue engineering is to create an artificial nerve graft that could mimic the extracellular matrix (ECM) and assist in nerve regeneration. Bio-composite nanofibrous scaffolds made from synthetic and natural polymeric blends provide suitable substrate for tissue engineering and it can be used as nerve guides eliminating the need of autologous nerve grafts. Nanotopography or orientation of the fibers within the scaffolds greatly influences the nerve cell morphology and outgrowth, and the alignment of the fibers ensures better contact guidance of the cells. In this study, poly (L-lactic acid)-co-poly(ε-caprolactone) or P(LLA-CL), collagen I and collagen III are utilized for the fabrication of nanofibers of different compositions and orientations (random and aligned) by electrospinning. The morphology, mechanical, physical, and chemical properties of the electrospun scaffolds along with their biocompatibility using C17.2 nerve stem cells are studied to identify the suitable material compositions and topography of the electrospun scaffolds required for peripheral nerve regeneration. Aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds with average diameter of 253 ± 102 nm were fabricated and characterized with a tensile strength of 11.59 ± 1.68 MPa. Cell proliferation studies showed 22% increase in cell proliferation on aligned P(LLA-CL)/collagen I/collagen III scaffolds compared with aligned pure P(LLA-CL) scaffolds. Results of our in vitro cell proliferation, cell-scaffold interaction, and neurofilament protein expression studies demonstrated that the electrospun aligned P(LLA-CL)/collagen I/collagen III nanofibrous scaffolds mimic more closely towards the ECM of nerve and have great potential as a substrate for accelerated regeneration of the nerve.

Perspectives in regeneration and tissue engineering of peripheral nerves

Peripheral nerve injury is a common casualty and although peripheral nerve fibers retain a considerable regeneration potential also in the adult, recovery is usually rather poor, especially in case of large nerve defects. The aim of this paper is to address the perspectives in regeneration and tissue engineering after peripheral nerve injury by reviewing the relevant experimental studies in animal models. After a brief overview of the morphological changes related to peripheral nerve injury and regeneration, the paper will address the evolution of peripheral nerve tissue engineering with special focus on transplantation strategies, from organs and tissues to cells and genes, that can be carried out, particularly in case of severe nerve lesions with substance loss. Finally, the need for integrated research which goes beyond therapeutic strategies based on single approaches is emphasized, and the importance of bringing together the various complimentary disciplines which can contribute to the definition of effective new strategies for regenerating the injured peripheral nerve is outlined.

<i>In Vitro</i> Evaluation of the Growth and Migration of Schwann Cells on Electrospun Silk Fibroin Nanofibers

Schwann cells (SCs) are primary structural and functional cells in peripheral nervous system and play a crucial role in peripheral nerve regeneration. Current challenge in peripheral nerve tissue engineering is to produce an implantable scaffold capable of bridging long nerve gaps and assist Scs in directing the growth of regenerating axons in nerve injury recovery. Electrospun silk fibroin nanofibers, fabricated for the cell culture in vitro, can provide such experiment support. Silk fibroin scaffolds (SFS) were fabricated with formic acid (FA), and the average fiber diameter was 305 ± 24 nm. The data from microscopic, immunohistochemical and scanning electron micrograph confirmed that the scaffold was beneficial to the adherence, proliferation and migration of SCs without exerting any significant cytotoxic effects on their phenotype. Thus, providing an experimental foundation accelerated the formation of bands of Bünger to enhance nerve regeneration. 305 nm SFS could be a candidate material for nerve tissue engineering.

PLGA conduit seeded with olfactory ensheathing cells for bridging sciatic nerve defect of rats

PLGA is thought to be a promising material for nerve scaffold. OECs have been shown to promote axon outgrowth and myelination following peripheral nerve transection. This study assessed the compatibility between PLGA and OECs in vitro, and evaluated the effect of PLGA conduit filled with OECs and extracellular matrix gel (ECM) (POE group) on 10 mm-defect sciatic nerve of rats. Silicon-OECs-EMC (SOE group), PLGA-ECM (PE group), and silicon-ECM (SE group)-were used as the controls. The survival and distribution of OECs in vivo, neurohistology and neurofunction of the bridged nerve, were quantitatively evaluated from 1 week to 12 weeks after surgery. PLGA possessed complete compatibility with OECs. After implantation, OECs migrated along the axis of the nerve and survived longer in the POE group than in the SOE group. Gross recovery of the animal, like ulcerious and autophagical rate as well as relative diameter recovery rate of the fiber, was more successful in the POE group than in other groups. The number of the fiber in the middle and distal segments of bridged sites and neurons in anterior horn of the spinal cord was increased in both OECs-contained groups, but the diameter and the myeline thickness of the fiber were increased only in the POE group. The nerve conduction velocity and the amplitude of compound muscle active potential were improved much successfully in the PLGA-guided group than in the silicon-guided group, but the best improvement was encountered in the POE group. Sciatic function index was not improved in all groups at 12 weeks after surgery due to the injury model. These results suggested that PLGA filled with OECs is a significant alternative to conventional autograft in repairing peripheral nerve defects, and OECs are potential seed cells for peripheral nerve tissue engineering.

PKH26 dye for tracing the seed cells of tissue-engineered peripheral nerve in vivo

Objective To assess the validity of PKH26 dye for labeling the bone marrow mesenchymal stem cells (BMSCs) and the feasibility of this method for tracing the seed cells of tissue engineering nerve in vivo. Methods BMSCs isolated from Wistar rat's bone marrow were labeled with the PKH26 dye, the marked validity was observed by the fluorescence microscopy and flow cytometer, the cellular vitality was de-tected by MTT, the differentiated abilities were tested by in vitro osteablastic and lipoblastic differentiation. After that, the labelld cells were micro-injected into the acellular nerve grafts(ANG) to build compound grafts and cultured for 3,5,7,14 days in vitro, then frozen sections were made respectively to observe the survival and migration of the implanted cells in ANG. Simultaneously, these compound grafts were used to bridge the 1.5 cm sciatic nerve defects of the Wistar rats, 1,4,6,8 weeks postoperation, the grafts were taken out and frozen section were made in order to observe the survival of the implanted stem cells in vivo inside the ANG. Results PKH26 dye was good for labelling the BMSCs, marked validity was higher than 95%. The cellular vitality and differentiated abilities were not affected compared with the unlabelled cells. Further more, BMSCs could adhere to the ANG, and suvive,magrate in vitro. In vivo, BMSCs was able to survive at least 8 weeks in the nerve regenerative microenvironment. Conclusion PKH26 dye could label the BMSCs effectively, and it's suitable for in vivo seed cells tracing in tissue engineering nerve model. Key words: Tissue engineering; Peripheral nerve; Bone marrow mesenchymal stem cells

Genetically modified canine Schwann cells—In vitro and in vivo evaluation of their suitability for peripheral nerve tissue engineering

After peripheral nerve injury, Schwann cells (SC) guarantee for a regeneration-promoting milieu and are crucially involved in axonal regeneration. For extended nerve defects, bridging with an autologous nerve transplant is the gold standard therapy. Artificial biohybrid nerve transplants which combine a synthetic conduit with autologous SC genetically modified to express regeneration-promoting proteins may provide an alternative therapy to autotransplantation. The dog seems to be an ideal translational animal model for new treatment strategies. In the present study, utilizing a new transfection protocol, we transplanted enhanced green fluorescent protein (EGFP)-expressing adult canine SC (cSC) into a 5 mm epineural pouch in the sciatic nerve of adult rats ( n = 9). The epineurial pouch technique serves as proof of principle to follow the fate of the transplanted cSC for up to 14 days after surgery. Fluorescence microscopy and immunohistochemistry revealed survival and integration of EGFP-expressing cSC into the regenerating host nerve tissue. We demonstrate that transplanted cSC contribute to the formation of bands of Büngner and are located in close vicinity to growth-associated protein-43 (GAP-43) expressing regenerating nerve fibers. This provides first evidence that transplanted genetically modified Schwann cells do successfully integrate into the host tissue where they could actively contribute to the regeneration process.

A new cell culture protocol for enrichment and genetic modification of adult canine Schwann cells suitable for peripheral nerve tissue engineering

Easily applicable techniques are presented to obtain high numbers of enriched canine Schwann cells (cSC) in a short time-window. The potential of adult SC for tissue engineering of peripheral nerves and ex vivo gene therapy is obvious from physiological events taking place after peripheral nerve transection [Haastert, K., Grothe, C., 2007. Gene therapy in peripheral nerve reconstruction approaches. Curr. Gene Ther. 7, 221–228]. The presented techniques were modified from a protocol for cultivation and expansion of adult cSC by others [Pauls, J., Nolte, C., Forterre, F., Brunnberg, L., 2004. Cultivation and expansion of canine Schwann cells using reexplantation. Berl. Munch. Tierarztl. Wochenschr. 117, 341–352] and own experiences in rodent and human SC cultivation and transfection [Haastert, K., Mauritz, C., Chaturvedi, S., Grothe, C., 2007. Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nat. Protoc. 2, 99–104]. A purity of about 80% cSC achieved by immunopanning techniques and selective culture conditions is 2.5 fold higher as previously reported ( Pauls et al., 2004). Additionally, highly enriched cSC populations are available in 3–4 weeks, only half the time period reported previously ( Pauls et al., 2004). Furthermore, electroporation and genetic modification of cSC is reported for the first time.