Tissue Engineering of Bone – Ch 13Tissue Engineering of the Liver – Ch 15

Culture of Neuroendocrine and Neuronal Cells for Tissue Engineering– Ch 14

Reference: Culture of Cells for Tissue Engineering (Culture of Specialized Cells), Chapter 13, 14 & 15

Shu-Ping Lin, Ph.D.

Date: 05.23.2011

Institute of Biomedical EngineeringE-mail: splin@dragon.nchu.edu.tw

Website: http://web.nchu.edu.tw/pweb/users/splin/

Tissue Engineering

Tissue Engineering of Bone – Ch 13

It is apparent that silk protein-based polymers are logical choices for biocompatible scaffolding for the formation of advanced matrices to induce bone tissue repair because of their mechanical properties and other advantages Silk Purification,

Preparation of Silk-RGD (arginine-glycine-aspartate), Preparation of Silk Scaffolds

Expression/activity of transcription factors, regulating lineage restriction, commitment, and/or differentiation within some of the mesenchymal lineages including osteoblasts

Regulatory transcription factor for osteogenic differentiation is cbfa1. Cbfa1 gene deletion results in complete absence of bone formation and mature osteoblasts;cbfa1 overexpression blocks osteoblast maturation

Stimulatory effect of BMPs may be the dependence of the actions of growth factors on the relative stage of differentiation of the target cells BMP-2 significant effects

on total mineralization were observed at concentrations as low as 40 ng/ml compared to medium without supplementation of BMP-2

Avoiding cost-intensive use of recombinant growth factors is the use of adenoviral transfection.

Glucocorticoids such as dexamethasone, even a transient exposure of stem cells to dexamethasone may be effective in inducing and maintaining the osteoblasticphenotype

Glycerophosphate such as L-Ascorbic acid (AA, vitamin C) increases cell viability and is a cofactor in the hydroxylation of proline and lysine residues and is therefore necessary for the production of collagen; prerequisite for the formation and mineralization of the extracellular matrix

Bone marrow contains at least two distinct populations of stem cells, one hematopoietic and the other nonhematopoietic mesenchymal.

Hematopoietic stem cells give rise to immune and blood system

MSC can differentiate into bone, cartilage, or adipose tissue

Adult bone marrow at an exceedingly low frequency of <1 MSC per 106 bone marrow cells, low frequency of mesenchymal cells within the marrow compartment has led investigators to develop techniques for stem cell isolation and culture.

Routine assessment can involve the analysis of the cell morphology, as mesenchymalcells are typically adherent marrow cells, generally considered to be spindle shaped.

Further analysis involves: surface antigen expression by flow cytometry; their ability to differentiate along skeletal lineages; the number of expansion cycles possible without a significant reduction in differentiation capacity (generally observe stable differentiation capacity up to 4 passages)

SH2 antibodies originally developed by Caplan and CD105, which is also known as endoglin and is a regulatory component of the TGF-β receptor complex.

Evaluating the absence of adherent and nonadherent cells, in particular of hematopoietic (CD34) or endothelial origin (CD31)

ISOLATION AND CULTURE METHODOLOGY OF HMSC

Schematic overview of

experimental approach

In addition to biological stimuli, bioreactors provide physiologically relevant physical signals (e.g., interstitial fluid flow, shear, pressure, mechanical compression) A recent study details the

geometry and extent of mineralized tissue under laminar and dynamic flow conditions

mRNA analysis: real-time quantitative RT-PCR assays for genes encoding for (i) osteoblast-related membrane and extracellular matrix molecules (i.e., alkaline phosphatase (ALP), aggrecan (Agg), bone sialoprotein (BSP), osteopontin (OP), type 1 collagen (Col1)); (ii) bone morphogenetic protein 2 (BMP-2), an important growth factor determining cell mesenchymal precursors; and (iii) cbfa1 (a genetic regulator of osteoblast function and also known as Osf2 or RUNX2), a transcription factor related to osteogenesis.

BMP-2 is a molecule attracting MSCs, and it induces proliferation and differentiation of mesenchymal progenitor cells, producing bone tissue even at ectopic sites

Osteopontin is one of the most abundant noncollagenous proteins in bone; it binds to various extracellular molecules, including type I collagen, fibronectin, and osteocalcin, and may add physical strength to extracellular matrices

A) phase-contrast photomicrographs of putative passage 2 MSCs at approximately 40% confluence at an original magnification of ×20. B) Surface CD105 expression of

passage 2 MSCs. C) Characterization of chondrocyte differentiation of MSCs in pellet culture, treated either with chondrogenic medium or with control medium (insert). Pellet diameter is approximately 2 mm, stained with safranin O. D) Characterization of osteoblast differentiation of MSCs in pellet culture, treated either with osteogenicmedium or with control medium (insert). Pellet diameter is approximately 2 mm, stained according to von Kossa. E) Sulfated GAG/DNA (µg/ng) deposition of passage 1 and 3 MSC pellet culture after 4 weeks. F) Calcium deposition/DNA (µg/ng) and AP activity/DNA (units/ng) of passage 1 and 3 MSC pellet culture.

Characterization of MSCs

Treat large bone defects after, for example, trauma or in the context of tumors do not

restore major damage to a tissue or organ in a truly satisfactory way, and the need for bone substitutes increases with the progressive aging of the population rely on the

harvest of autologous bone, a method limited by the availability of transplant material, harvesting difficulties, donor site morbidity, additional pain, longer hospital stays, and higher treatment costs, with a direct impact on function, availability, and psychological and social well-being of patients.

Novel methods currently being studied include conduction (by a scaffold) and induction (by bioactive molecules) of cell migration to repair relatively small defects and cell transplantation into the defect site (with or without biomaterial) to repair larger defects

Did not observe this orientation of the deposited bone in faster degrading collagen protein or on PLGA composite scaffolds. Therefore, silks can provide a blueprint for the desired bone geometry through the design of scaffold structures.

Geometries less than 200 µ m result in the formation of a dense and continuous bone plate, originating from a coalescing trabecular network

Complex composite tissues within a single scaffold requires the exposure of one cell source—mesenchymal stem cells—to different growth factors to drive the differentiation along the desired lineages: Silks provide all kinds of functional groups, allowing covalent decoration through easy chemical reaction.

IN VITRO APPLICATIONS

Tissue engineering of bone-like tissue. Bar = 5 mm (A). Magnification shows trabecula-like structure (B).

Tissue Engineering of the Liver – Ch 15

Liver failure is a significant clinical problem, representing the cause of death of over 30,000 patients in the US every year and over 2 million patients worldwide.

Organ transplantation is the only therapy to date shown to alter mortality, although its utility is restricted because of the scarcity of donor organs.

Partial liver transplants from cadaveric or living donors have been demonstrated to be effective treatments and a means to increase the supply of donor organs

body’s role in the regulation of liver mass and the significant capacity for regeneration exhibited by the mammalian liver; partial hepatectomy or chemical injury induces the proliferation of the existing mature cell populations within the liver including hepatocytes, bile duct epithelial cells, and others, resulting in the replacement of lost liver mass.

Liver regeneration is not a component of all disease settings (e.g., cirrhosis) and is difficult to control clinically

Alternative approaches include nonbiological extracorporeal support systems, such as plasma exchange, plasmapheresis, hemodialysis, or hemoperfusion over charcoal or various resins, although these systems have achieved limited success.

Cell-based therapies: Liver exhibits over 500 functions, including detoxification, synthetic, and metabolic processes recapitulation of a substantial number of

liver functions include the transplantation of hepatocytes, perfusion of blood through an extracorporeal device containing hepatocytes, transgenic xenografts, or the implantation of hepatocellular constructs

Liver Disease and Cell-Based Therapies

Cell Sources for Liver Cell-Based Therapies

Cell-based therapies for liver disease. Extracorporeal devices perfuse patient’s blood or plasma through bioreactors containing hepatocytes. Hepatocytes are transplanted directly or implanted on scaffolds. Transgenic animals are being raised in order to reduce complement- mediated damage of the endothelium.

Immortalized hepatocyte cell lines such as HepG2 (human hepatoblastoma) or HepLiu (SV40 immortalized) but display

an abnormal assortment of differentiated functions and for clinical applications there is a risk that oncogenic factors could be transmitted to the patient.

Various stem cell populations (embryonic and adult), which retain significantproliferative ability in vitro and exhibit pluripotency (embryonic) or multipotency (adult), thereby providing a potential source of hepatocytes as well as other liver cell types (bile duct epithelial cells from a single proliferative precursor, represents a potential means for enhancing liver-specific function of a tissue engineered construct.

Challenges: the ability to control the microenvironment and organize resultant structures, strategies for delivery, stabilization of hepatocyte functions, etc.

Promote hepatocyte stabilization in vitro, with a goal of mimicking physiologically relevant extracellular cues that are absent from standard culture models

modifications in culture medium such as hormonally defined preparations with low concentrations of hormones, corticosteroids, cytokines, vitamins, or amino acids, or the addition of low levels of dimethyl sulfoxide or dexamethasone

Extracellular matrix of various compositions is also known to exhibit positive effects on hepatocyte function: sandwich culture of hepatocytes within collagen gel or culture on the tumor-derived basement membrane preparation Matrigel(formation of spheroid cellular structurescell-cell communication homotypic

(hepatocyte-hepatocyte) interactions, spheroidal aggregates have been shown to promote the formation of bile canaliculi, gap junctions, and tight junctions and the expression of E-cadherins and several differentiated functions)

Heterotypic (hepatocyte-nonparenchymal) interactions, hepatocytecocultures with both liver- and non-liver-derived cell types, have also been shown to improve viability and differentiated function. But potential regulatory factors, such as secreted signals (i.e., cytokines) or cell-associated signals (i.e., insoluble extracellular matrix or membrane-bound molecules), in the ―coculture effect‖ have not yet been clearly defined.

Bioreactor designs: Perfusion systems facilitate nutrient delivery to hepatocytes, are highly metabolic in contrast to a batch process, would enable the continuous processing of blood or serum, potentially useful for extracorporeal devices.

Approaches for the In Vitro Stabilization of Primary Hepatocytes

The majority of liver cell-based bioreactor designs fall into these four general categories: flat plate, hollow fiber, perfusion scaffolds, and packed beds, each with inherent advantages and disadvantages

Combination of a flat plate reactor system with sandwich culture or coculture has been illustrated to improve hepatocyte stability for long-term analysis

Three-dimensional perfusion bioreactor system has been developed based on morphogenesis of hepatocytes into 3D structures in an array of channels

Similar to static culture models, preaggregation of hepatocytes into spheroids enhanced the functionality of hepatocytes in this design, underscored the importance of microenvironmental signals, including soluble mediators, cell-extracellular matrix interactions, and cell-cell interactions, in the regulation of hepatocyte processes.

Schematics of cell-based bioreactor designs

Extracellular signals in hepatocyte culture in vitro correlates with the significant role of the microenvironment in the liver in vivo.

Complex combination of extracellular cues, including tightly controlled cell-cell interactions and distribution of extracellular matrix

Liver microenvironment such as fibrosis and have additionally been implicated in repair processes

A crucial issue for not only the improvement of in vitro culture models and extracorporeal liver devices, but also the manufacture of implantable hepatocyteconstructs represent ideal platforms for ADMET (adsorption, distribution,

metabolism, excretion, and toxicity) testing of novel drug candidates

Strategy toward the fabrication of engineered liver tissue is to develop (1) enabling platforms for the controlled recapitulation of these critical extracellular cues and (2) in vitro models to study the complexities of environmental interactions

Important criteria for the development of a particular system include (1) the desired application (in vitro model, basic science, or therapeutic) and (2) the important liver properties that must be established for that certain application, such as stabilized phenotype, structural complexity (zonation), directional fluid flow, and in some cases 3-D organization.

Investigate systematically the role of heterotypic contact in hepatocytecocultures developed and characterized a versatile micropatterning method, which has enabled the quantitative control of the spatial relationship of hepatocytesand fibroblasts; such as continuous signaling, gap junctions, and soluble factors

Regulation of the Hepatocyte Microenvironment Toward the Development of Engineered Liver Tissue

Several parameters are utilized when assessing primary hepatocytestabilization in vitro:

Classic hepatocyte morphology including a cuboidal shape and well-defined cell borders with intact bile canaliculi is considered suggestive of a mature stable phenotype.

Four categories of liver function: metabolism, synthesis, bile excretion, and detoxification (phase I and phase II). Such as: albumin secretion (ELISA, immunofluorescence or immunohistochemistry for detection of interacellular albumin), urea synthesis, and cytochrome P-450 (CYP) (phase I) enzyme activity, a potentially effective bioartificial liver device, assays for bile duct excretion and phase II activity, the degree of hepatocyte stabilization

Microarray analysis of the expression of a broad range of functionally important genes can be utilized.

Measurement of hepatocyte-specific functions is important in the assessment of hepatocyte differentiation from stem cell sources such as embryonic stem cells or various adult stem cell populations

ASSAYS OF HEPATOCYTE FUNCTION

Cell-cell and cell-extracellular matrix interactions: critical for proper cellular processes in numerous organ systems and key determinants of hepatocyte function.

Isolated hepatocytes cultured in monolayer culture display a rapid loss of phenotype, preventing the effectiveness of these cells in long-term applications

Cell-cell interactions, both homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-nonparenchymal), have been demonstrated to exhibit positive effects on hepatocyte function.

Hepatocyte viability and a variety of liver functions have been shown to be stabilized for weeks in vitro on cocultivation with liver-derived cell types as well as some non-liver-derived populations

The role of cell-cell interactions in hepatocellular function, developed and characterized a flexible method of micropatterning cocultures of hepatocytes and an embryonic fibroblast cell line (3T3-J2) on solid substrates (U.S.PatentNo. 6,133,030): photolithographic techniques used to pattern collagen I on glass substrates, which served as an adhesive substrate for primary rat hepatocytes. After hepatocyteattachment and spreading, 3T3-J2 fibroblasts were plated, by serum-mediated adhesion, in the remaining intermixed regions. Total cell numbers were held constant by holding surface area of each domain (collagen I/bare glass) equal for each condition. Liver functions, including albumin secretion and urea synthesis, were increased in cocultured configurations compared to hepatocytes, upregulation varied with culture configuration cocultures with a larger initial heterotypic interface (i.e., single-cell islands) exhibited increased levels of liver-specific function

MICROPATTERNED CELL CULTURES

Phase-contrast micrographs of micropatternedcocultures indicate a broad range of heterotypic interface achieved despite similar cellular constituents. Four of five patterns used in study are shown. Diameters of hepatocyte islands were 36 µm (A), 100 µm (B), 490 µm (C), and 6800 µm (D).

Micropatterned cocultures with constant ratio of cell populations

Liver-specific function of

micropatternedcocultures with

constant ratio of cell populations

Urea synthesis (A) and albumin secretion (B) on day 11 of culture were detected in micropatterned cocultures with varying heterotypic interactions despite similar cell numbers. Micropatterned hepatocyte-only cultures were utilized as a control. Statistical significance (*) was determined by one-way ANOVA with Tukey HSD post hoc analysis with P <0.05.

In situ markers are equally important, particularly for the analysis of the role of the microenvironment on individual hepatocyte function. Immunohistochemicalstaining indicated that hepatocytes near the heterotypic interface had a relative increase in liver-specific function, bulk tissue function (increased heterotypic interactions equaled increased function): utilization of micropatterningtechniques enabled a 12-fold reduction in fibroblast numbers with only a 50% reduction in albumin secretion, due to the ability to control the extent of heterotypic interactions. Reduction in the required numbers of nonparenchymal cells, which would occupy precious substrate surface area within a potential bioreactor, would essentially increase hepatocyte functionality per unit area. Thus micropatterningapproaches would allow for significant improvements in the design of a projected coculture-based bioreactor.

Intracellular albumin in micropatterned hepatocytes. Immunohistochemicalstaining of intracellular albumin in micropatterned hepatocytes in a representative (490 µm) pattern. Bright-field microscopy of hepatocytes (alone) on days 1 and 6 (A, C) and cocultures on days 1 and 6 (B, D). In coculture, albumin expression was highest near the heterotypic interface.

Schematic of method for generating micropatterned

cocultures Borosilicate substrates were coated with

photoresist (a UV-sensitive polymer) and exposed to light through a mask, creating a photoresist pattern (A). Photoresist was visualized with epifluorescence microscopy (B) (excitation: 550 nm, emission: 575 nm). Collagen I was immobilized, followed by removal of photoresist, yielding a collagen-glass pattern (C). Indirect immunofluorescence allowed verification of collagen immobilization in appropriate locations (D). Patterned substrates were exposed to hepatocytes in serum-free media and rinsed, resulting in micropatterned hepatocytes (E). (F) Phase-contrast micrograph of 200 um lanes of hepatocytes with 500 um lane spacing. Addition of 3T3-J2 fibroblasts in medium supplemented with serum resulted in generation of micropatternedcocultures (G). Phase-contrast microscopy allowed morphological identification of 2 distinct cell types in ―micropatternedcoculture‖ (H).

Potential problems include peeling of photoresist during processing and difficulty with lift-off of photoresist integrity of the photoresist coating varies with solvents Photoresist will peel away from the wafer surface prematurely. This

indicates either insufficient adhesion of the photoresist to glass (often because of an insufficient dehydration bake before photoresist coating) or insufficient baking after development to harden the photoresist. Difficulty with photoresist removal resulting in extended sonication in acetone often indicates exposure of photoresist to elevated temperatures.

Cellular Micropatterning on Modified Surfaces: localized modifications of surface chemistry to pattern cells by patterning adhesive molecules including covalent coupling of proteins to the surface by a modified technique as well as direct adsorption of proteins from solution utilized for the micropatterning of

primary hepatocytes (attach specifically to the collagen I patterned regions) and an embryonic fibroblast cell line (undergo nonspecific, serummediated attachment to the remaining unmodified areas)

Collagen type I retained its bioactivity for hepatocyte attachment and spreading despite treatment with acetone, ethanol, and dehydration. Other proteins may require modifications of this protocol to retain their bioactivity.

Because of the rapid mitotic rate of fibroblasts, medium contains hydrocortisone as a growth inhibitor (under some circumstances fibroblasts can be chemically growth arrested with mitomycin C)

Other potential problems: lack of cell adhesion to the substrate, contaminants that coat the glass and prevent protein immobilization, lack of discernible pattern because of exuberant cell adhesion

BIOREACTOR SYSTEM FOR THE REGULATION OF HEPATOCYTE ZONAL HETEROGENEITY

Numerous hepatocyte functions are known to vary along the length of the liver sinusoids from the portal triad (i.e., portal vein and hepatic artery) to the central vein, a feature termed ―liver zonation‖; urea synthesis and gluconeogenesis are localized near the portal triad (periportal region), processes of cytochrome P450 activation and detoxification, as well as glycolysis, are enriched near central vein (perivenousregion).

Zonated features along the sinusoid. The metabolic activity of hepatocytes along the liver sinusoid creates oxygen and hormone gradients from the periportal region to the perivenous. As a result, metabolic and detoxification functions are regionally dominant to one zone or the other. Some specific zonated markers are listed under each process. Abbreviations: HA, hepatic artery; PV, portal vein; CV, central vein; PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; FBPase, fructose-1,6-bisphosphatase; GK, glucokinase;

PK, pyruvate kinase; CYP, cytochrome P-450.

Bioreactors were operated with an inlet pO2 of 76 and 158 mmHg and flow rate of 0.5 ml/min. The resulting cell surface oxygen gradients are shown schematically as calculated from the numerical model (A). Western blots of PEPCK (B) and CYP2B (C) protein levels from four regions along the chamber were analyzed to determine relative optical density. In both cases, when the bioreactor was operated with physiologic gradient (low inlet), a heterogeneous induction was observed, whereas imposing a supraphysiologic gradient (control, high inlet) resulted in a more uniform protein distribution. Blots were processed in separate experiments, enabling only qualitative comparison between conditions. Normalization of band densities to the maximal density from both experiments is meant to facilitate comparison.

Heterogeneous induction of PEPCK and CYP2B by oxygen

gradients

Schematic of bioreactor circuit Parallel-plate bioreactor system

containing cultured hepatocytes results in the formation of steady-state O2 gradients, generating zonated hepatocyte phenotypes.

O2 concentration profile can be modeled as a combination of O2 diffusion to the cell, uptake by the cell, and convection along the length of the flow region.

Inlet O2 concentration representative of physiologic periportal levels, 76 mmHg (10% O2), a flow rate of 0.5 ml/min was determined to result in the exposure of cells in the final 50% of the chamber length to perivenous O2 levels (<35 mmHg). O2

distribution resulted in zonated hepatocytephenotypes within the bioreactor.

Shear stress experienced by the cultured cells flow rate of 0.5 ml/min corresponds to 1.25 dyn/cm2, well below the shear stress of 5 dyn/cm2, and under O2 inlet of 76 mmHg perfect for preventing cellular damage due to shear stress or hypoxia

Poly(ethylene glycol) (PEG)-based hydrogels are widely used in tissue engineering applications because of their biocompatibility and hydrophilicity resulting in resistance to nonspecific protein adsorption and the ability to alter the properties of the hydrogel by modifying the PEG chain length

Hydrogel microstructures containing living cells. A) Cells entrapped in PEGDA hydrogels patterned in various shapes. B) Red and green labeled cells patterned within distinct domains of a single hydrogel layer. C) Phase microscopy of cellular array covalently linked to glass substrate. D) Fluorescent image of the cellular array, showing two different cell types (green, red).

PHOTOPATTERNED THREE-DIMENSIONAL HYDROGELS CONTAINING LIVING CELLS

Multilayer hydrogel microstructures containing living cells

A) Two layers of patterned PEGDA lines. B) Two layers of patterned PEGDA lines containing cells. C) Fluorescent image of patterned hydrogel lines containing red and green tracked cells, demonstrating different cells in each layer. D) Three layers of patterned PEGDA lines containing cells at low magnification. E) Three layers of patterned PEGDA lines containing cells at higher magnification. F) Fluorescent image of three layers, with one layer containing red labeled cells, one layer containing green labeled cells, and all layers counterstained blue.

Process for formation of hydrogel microstructures containing living cells. The apparatus is assembled, including a pretreated glass wafer with reactive methacrylate groups on its surface, and a Teflon base with an inlet and outlet. Once the cells and prepolymer solution are injected, the inlet and outlet are closed, and the unit is exposed to UV light. The resulting patterned hydrogels containing cells are covalently bound to the glass wafer. At this time, a thicker spacer can be used in conjunction with a new mask to add another layer of cells. This process can be repeated several times.

Overall approach for the design of engineered liver tissue. Each of these aspects will provide fundamental insight into liver biology and constitute important components contributing to the future development of highly functional tissue-engineered liver constructs.

Culture of Neuroendocrine and Neuronal Cells for Tissue Engineering–

Ch 14

Motivation

59% of all admitted military patients who have been exposed to a blast have been diagnosed with traumatic brain injury (TBI)

During one year of conflict, 437 cases of TBI were diagnosed at

Walter Reed Army Medical Center alone

Over half of these patients had permanent brain damage.

In the U.S. civilian population, there are 1.4 million new cases of TBI each year, with $60 billion in healthcare expenses.

Today, 5.3 million Americans live with TBI.

The Stem Cell Concept

Stem cells are defined as pluripotent, self renewing cells.

Recent discoveries have found that certain populations of adult stem cells are capable of transdifferentiating to generate cells in a different germinal layer. Non-Hematopoietic SCs:

skeletal muscle

cardiac muscle

liver

Neural SCs: blood cells

skeletal muscle

Bone marrow SCs: cardiac muscle

skeletal muscle

neural cells

Previous Studies

In vivo

In vitro

Reh criteria – A functional, mature neuron is:

Post–mitotic

Polarized with a single axon, multiple dendrites

Able to fire action potentials

Able to communicate with other neurons

Differentiation Methods

Beta-mercaptoethanol (Woodbury et. al.) Critique: Toxicity and aberrant marker expression

Two step neurosphere differentiation (Hermann et al.) Convincing comparative analysis of multiple methods showed a two

step differentiation process to be promising, but time consuming.

Retinoic Acid (Cho et al.) Morphological, phenotypic and functional analyses looked

convincing

Astrocyte co-culture (Bossolasco et al., Song et al.) Theory and evidence behind study looked encouraging

Neuron-specific Markers

Marker Function Type

Nestin Neural intermediary filament type IV. It is an important cytoskeletal component of neuronal axons and seems to have an influence over axonal diameter.

neural progenitor marker (early)

β-tubulin III Microtubule subunits in neurons Immature neuronal phenotype

NeuN Soluble nuclear protein that binds to neural DNA. It is exclusive to tissue in the nervous system, and is only expressed by the neurons in the nervous system.

Intermediate neuronal phenotype

MAP2ab Neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro.

Late neuronal phenotype

GFAP Type III protein of the intermediate filament exclusively found in astrocytic cells of the central nervous system

Glial phenotype

synaptophysin A calcium-binding glycoprotein that is present in the presynaptic vesicles of neurons and in the neurosecretory granules of neuroendocrine cells.

Synaptic transmission

Objective

Establish and optimize protocols for characterizing the differentiation of MSC samples using neural progenitors.

Compare different neural induction methods

Investigate the effects of utilizing two different scaffold types

Big Picture

Cells Scaffold

Signals

Stem Cell Delivery Device for Neural Injury

Materials

Cells Neural progenitors (NPs) Normal human astrocytes (NHAs) Human bone-marrow-derived mesenchymal stem cells (MSCs)

Retinoic acid (RA) An essential stimulus of cell growth and differentiation, especially in

embryos

Matrices Collagen

Provides cell affinity Biocompatible Poor mechanical properties

Chitosan Provides anti-infection properties Stops hemorrhaging, promotes wound healing Stronger mechanical properties

Methods - Differentiation

Treatment with retinoic acid (RA) alone Two week timeframe

Feedings 3-4 days with RA media at low concentration

Monolayer and 3D

Treatment by MSC + astrocyte co-culture Three week timeframe

Feedings 3-4 days with 50/50 MSC and Astrocyte media

Monolayer and 3D

Treatment with both RA and Astrocyte co-culture Two week timeframe

Monolayer

Methods - Characterization

Light microscopy

Samples viewed 3-4 days prior to feeding

Confocal

RA and RA co-culture samples fixed at 7 and 14 day time points.

Astrocyte co-culture samples fixed at 14 and 21 day time points.

SEM

With the same time points above

Results: Untreated Mesenchymal Stem Cells

Nestin: intermediate filament protein expressed in neuronal precursor cells.

Result: MSCs express nestin!

Questions:

Can MSCs differentiate into neurons?

What is the best method of differentiation?

Green: Nuclei Red: Nestin

Method 1: RA-treated MSCs (Day 6)

MSCs before RA-treatment MSCs exposed to RA for 6 days

Results:

Within 6 days…

• Cell bodies became increasingly spherical and refractile

• Long, thin processes extend from cell bodies

Method 1: RA-treated MSCs (Day 8)

Results:

Within 8 days…

• Monolayer: high btubulinIII and MAP2

• Collagen: high btubulinIII, low MAP2

• Chitosan: low btubulinIII, MAP2 inconclusive

Monolayer Collagen

Green/yellow: nuclei

Blue: MAP2

Red: btubulin III

Pink: colocation

Chitosan

Method 1: RA-treated MSCs (Day 14)

Day 14

Results:

Within 14 days, RA-treated MSCs exhibit bipolar neuronal morphology with axons

Method 1: RA-treated MSCs (Day 14)

20X 10X 10X

Monolayer of RA-treated MSCs after 14 days

Results:

• High btubulinIII expression

• Very low MAP2 expression

• Dim, scattered nuclear staining Unhealthy cells?

Green/yellow: nuclei

Blue: MAP2

Red: btubulin III

Pink: colocation

Method 2: MSC + Astrocyte (Day 14)

Monolayer Collagen Chitosan

Results:

• Monolayer: High btubulinIII, low MAP2

• Collagen: High btubulinIII and MAP2

• Chitosan: High btubulinIII, MAP2 inconclusive

Green/yellow: nuclei

Blue: MAP2

Red: btubulin III

Pink: colocation

Method 2: MSC + Astrocyte (Day 21)

Collagen Chitosan

Results:

• Monolayer: contamination

• Collagen: btubulinIII and MAP2 positive

• Chitosan: btubulinIII positive, MAP2 inconclusive

Green/yellow: nuclei

Blue: MAP2

Red: btubulin III

Pink: colocation

Method 3: RA-treated MSC + Astrocyte (Day 8 + 14)

Day 8 Day 14

Day 8 Day 14

Results:

• Day 8: high btubulinIII and MAP2

• Day 14: high btubulinIII, low MAP2

• Dim, scattered nuclear staining Unhealthy cells?

Method 3: RA-treated MSC + Astrocyte (Day 14)

MSCs + Astrocytes + RA Day 1 MSCs + Astrocytes + RA Day 14

Results:

Within 14 days…• Cell bodies became increasingly spherical and refractile• Long, thin processes extend from cell bodies

Day 14

Method 3: RA-treated MSC + Astrocyte (Day 14)

Results:

Within 14 days, RA-treated MSCs exhibit bipolar neuronal morphology with axons

Results: Neural Progenitors

Day 1 Day 5 Day 9

Day 11 Day 14 Day 16

Neurospheres…

…dendritic outgrowths and long processes

Collagen Chitosan

Results: Neural Progenitors

Results:

• monolayer: high btubulinIII and MAP2

• collagen: high btubulinIII and MAP2

• chitosan: btubulinIII, MAP2 inconclusive due to autofluorescence

Monolayer

Day 3 Day 8 Day 14 Day 21

RA monolayer Neurosphere morphology

B-tubulinIII

MAP2

B-tubulinIII

MAP2

*axons

N/A

RA collagen B-tubulinIII

MAP2 (low)

contamination N/A

RA chitosan B-tubulinIII

MAP2 inconclusive

contamination N/A

RA/astrocyte

monolayer

B-tubulinIII

MAP2

B-tubulinIII

MAP2

*axons

N/A

Astrocyte monolayer

N/A B-tubulinIII

MAP2

overconfluent

Astrocyte collagen

N/A B-tubulinIII

MAP2

B-tubulinIII MAP2

Astrocyte chitosan

N/A B-tubulinIII

MAP2 inconclusive

B-tubulinIII

MAP2 inconclusive

Comparison of Methods 1, 2, and 3

Future Studies

Functionality: Action potential

Patch clamp technique

Synaptic communication Synaptophysin: calcium-binding glycoprotein that is present in

the presynaptic vesicles of neurons

Scaffold: Collagen/chitosan composite hydrogel

In vivo testing MSCs alone? Pre-differentiated MSCs + factors? Differentiated MSCs?