Wednesday, December 27, 2006

 

Scientists create 3-D scaffold for growing stem cells

Stem cells grew, multiplied and differentiated into brain cells on a new three-dimensional scaffold of tiny protein fragments designed to be more like a living body than any other cell culture system.

An MIT engineer and Italian colleagues will report the invention - which may one day replace the ubiquitous Petri dish for growing cells - in the December 27th (2006) issue of the journal PLoS ONE [1]. Shuguang Zhang*, associate director of MIT's Center for Biomedical Engineering, is a pioneer in coaxing tiny fragments of amino acids called self-assembling peptides to organize themselves into useful structures. Working with visiting graduate student Fabrizio Gelain from Milan, Zhang created a designer scaffold from a network of protein nanofibers, each 5,000 times thinner than a human hair and containing pores up to 20,000 times smaller than the eye of a needle.

The researchers were able to grow a healthy colony of adult mouse stem cells on the three-dimensional scaffold without the drawbacks of two-dimensional systems.

In addition to helping researchers get a more accurate picture of how cells grow and behave in the body, the new synthetic structure can provide a more conducive microenvironment for tissue cell cultures and tissues used in regenerative medicine, such as skin grafts or neurons to replace brain cells lost to injury or disease.

The scaffold itself can be transplanted directly into the body with no ill effects.

Continued at "Scientists create 3-D scaffold for growing stem cells"

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[1] Excerpts from:

Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

Citation: Gelain F, Bottai D, Vescovi A, Zhang S (2006) Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures. PLoS ONE 1(1): e119. doi:10.1371/journal.pone.0000119

Abstract

Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with beta-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology.

Introduction

Nearly all tissue cells are embedded in a 3-dimensional (3-D) microenvironment in the body. However, almost all tissue cells have been studied in 2-D Petri dishes, 2-D multi-well plates or 2-D glass slides coated with various substrata. The architecture of the in situ environment of a cell in a living organism is 3-D, where cells are surrounded by other cells as well as many extracellular ligands, including many types of collagens, laminin, and other matrix proteins. The normal three-dimensional environment of cells comprises a complex network of extracellular matrix nanoscale fibers with nanopores that create various local microenvironments. These environments not only allow attachments between cells and the basal membrane, but also allow access to oxygen, hormones and nutrients, as well as removal of waste products.

The movements of cells in the 3-D environment of a living organism typically follow a chemical signal or molecular gradient, which is crucial for organism development. It is known that cells isolated directly from higher organisms frequently alter their metabolism and gene expression patterns in 2-D culture. Cells growing in a 2-D environment may significantly reduce production of particular extracellular matrix proteins and often undergo morphological changes, for instance, an increase in spreading.

Conventional 2-D cell cultures are unlike in vivo systems where cellular communication, transport of oxygen and nutrients, removal of wastes and cellular metabolism take place in a 3-D environment.

Attempts have been made to culture cells in 3-D using synthetic polymers and their copolymers [1]. However, many processed synthetic polymers consist of microfibers approx 10-50 micrometers in diameter, which are similar in size to most cells (approx 5-10 micrometers in diameter). Thus, cells attached to microfibers are still in a two-dimensional environment with a curvature dependent on the diameter of the microfibers. Furthermore, the pores (approx 10-200 micrometers) between the microfibers are often approx 1,000-10,000 times larger than the size of biomolecules, which have sizes just a few nanometers, including small molecular hormones, proteins, growth and other factors, which consequently can quickly diffuse away. For a true 3-D environment, a scaffold's fibers and pores must be substantially smaller than the cells. Although synthetic biopolymer microfiber scaffolds have been studied for over 30 years to mimic in vivo 3D microenvironment, concerns about their degradation products and chemicals involved in their synthesis are still important issues requiring further improvements.

Animal derived biomaterials such as collagen gels, laminin, poly-glycosaminoglycans and materials from basement membranes, including Matrigel have also widely been used in cell cultures [2]-[9]. While they are representative of the correct nanolength scale, they often contain residual growth factors, undefined constituents or non-quantified substances [2]-[6]. This not only makes it difficult to conduct well-controlled studies with these materials, but also poses problems if such scaffolds are ever to be used for growing tissues for human therapies.

An ideal 3-D cell culture system should be fabricated from a synthetic biological material with defined constituents. We previously reported the discovery of a self-assembling peptide system, made from natural amino acids, that can undergo spontaneous assembly into nanofiber scaffolds, approx 10 nm in fiber diameter with pores between 5-200 nm [10]-[12]. These peptides have been chemically produced in large quantity using standard solid phase synthesis method and purified to homogeneity. They have not only been used for the study of cell attachment, survival and proliferation but also to incorporate other motifs [11]-[16], and inject into animals [17]-[19]. These self-assembling peptides form nanofibers that can be controlled at physiological pH by altering salt concentration [10]-[11]. Because the self-assembled nanofibers are several thousand times thinner than synthetic polymer microfibers and cells, thus it is believed that the peptide nanofibers surround cells in a manner similar to the natural extracellular matrix. However a systematic study of different motifs and an examination of how their nanofiber scaffolds interact with cells in details have not been carried out.

Here we report the use of designer peptide nanofiber scaffolds to produce 3-D cultures for the study of mouse adult neural stem cells. We synthesized 18 different peptides that directly incorporate various functional motifs with the self-assembling peptide RADA16. These motifs include sequences shown to promote cell adhesion, differentiation and bone marrow homing activities. These functionalized peptides self-assemble into nanofiber scaffolds where cells can be fully embedded by the scaffold in 3-D. Without addition of soluble growth factors and neurotrophic factors, two of these scaffolds functionalized with bone marrow homing motifs [20] not only significantly enhanced survival of the neural stem cells, but also promoted differentiation towards cells expressing neuronal and glial markers. This is the first example suggesting that designer peptide scaffolds alone without additional extra growth factors could influence neural stem cell differentiation towards neural and glial phenotypes.

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*Shuguang Zhang "...discovered a self-assembling peptide system while working in molecular and structural biology with Alexander Rich at MIT. This serendipitous discovery was selected to be one of the fifteen research achievements over last quart century at MIT. He pursues actively on the various self-assembling peptide systems to develop a new class of biological materials including peptide matrix scaffold for tissue engineering, biological surface engineering for cell pattern formation, molecular switch, biological operating systems and surfactant peptide nanotubes. He also works on problems to gain understanding of a class of protein conformational diseases, including Alzheimer's, Parkinson's and the prion diseases (mad cow disease)..."

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