stem cell research

meets nanotechnology
Ricardo Pires das Neves 1,2 e Lino Ferreira 1,2
SUMMARY
The recent application of nanotechnolo-

1 CNC - Center of Neurosciences and Cell Biology, University of Coimbra, Portugal

gies into the stem cell field promises to

2 Biocant - Center of Innovation in Biotechnology, Cantanhede, Portugal

open new avenues in regenerative medicine. Nanotechnologies can be a valu-

Corresponding Author Contact: lino@biocant.pt

able tool to track and image stem cells,

INTRODUCTION

and other nanomaterials with sizes down to

stand their biology. This will hopefully

The existence of a multipotent hematopoi-

10 nm. In 1959, Richard Feynman launched

lead to stem cell-based therapeutics for

etic stem cell was demonstrated for the first

the foundation of the nanotechnology field.

the prevention, diagnosis, and treatment

time by Till and McCulloch in 1961. They

Since then, several extraordinary discover-

of human diseases. Despite these op-

demonstrated that a single hematopoietic

ies have been made: Richard Smalley dis-

portunities, nanotechnologies also pose

stem cell could (i) give rise to a mixed popu-

covered fullerenes in 1985, Sumio Iijima

several risks since they can be cytotoxic

lation of blood cells (granulocytes, macro-

discovered carbon nanotubes in 1991, and

and affect the differentiation program of

phages, red blood cells, etc) and (ii) had

Louis Brus the quantum dots in 1996.

stem cells. Here, we discuss the future

the ability to self-renew [1]. The isolation

The intersection of nanotechnologies with

opportunities and challenges that face

of mouse embryonic stem cells by Martin

stem cell research is recent and has been re-

this young field of research.

Evans in 1981, human embryonic stem cells

viewed by us elsewhere [2, 3]. In this work we

by James Thompson in 1998, and inducible

will review the current research topics in this

pluripotent stem cells by Shinya Yamanaka

area: stem cell microenvironment and tissue

in 2006, propelled the scientific community

engineering, stem cell tracking and imaging,

to understand the properties of these cells

stem cell transfection, isolation and sorting,

and evaluate their therapeutic effect in the

and molecular detection (Fig. 1). When ap-

context of the regenerative medicine.

propriate, we will describe some examples

The first observation of nanomaterials was

about the research that we are conducting

made by Richard Adolf Asigmondy in 1914.

at Centre for Neuroscience and Cell Biology

He performed a detailed study of gold sols

(CNC) and Biocant in this research area.

Figure 1. Nanotechnology applications in Stem Cell Biology and Medicine. Nanodevices can be used in stem cell tracking and imaging but also
in isolation and sorting of stem celis, both for basic science and translational medicine. Stem cell fate can be modulated by internalization
of nanocarriers with biological molecules or by external cues given by biomimetic scaffolds. Stem cell transfection and molecular detection
make use of nanodevices for intracellular access but also for intelligent delivery and sensing of biomolecules. These technologies have a
great impact in stem cell microenvironment and tissue engineering studies and have a great potential for biomedical applications.

38.

canalBQ_n. 7_DEZEMBRO_2010

Modulation of stem cell-fate by the microen-

the following aspects: (i) Laser fabricated

vironment

nanogrooves to study cell-cell interactions;

Stem-cell biology has been studied mainly

(ii) nanowires to study intra- and intercel-

in vitro with cells cultured on flat substrates

lular biological processes; (iii) nanophase

coated, for example, with collagen or lam-

thin film to study cell adhesion and pro-

inin, or in co-culture systems where feed-

liferation; (iv) Lab-on-a-chip with nano-

er-cell layers are used to support stem cell

reservoir to study environmental cues; (v)

ters in a given tissue is critical in deter-

growth. These culture conditions are very

Self-assembly peptides and nanofibers to

mining how cells behave within that tissue.

different from the environment that stem

mimic ECM; (vi) Nanoliter-scale synthesis

The ECM can be reproduced in vitro by the

cells experience in the body. For example,

of arrayed biomaterials; (vii) Micro/nanopat-

use of 3D scaffolds. For that purpose, sev-

the extracellular matrix (ECM) is difficult to

terned surface to study stem cell response

eral natural (fibrin, collagen, hyaluronic

mimic in plastic dishes; most frequently

to topography and mechano-transduction;

acid, etc) or synthetic (polyethylene

stem cells are cultured in rigid polystyrene

and (viii) Nanoparticles to control release

glycol, poly(lactic acid-co-glycolic acid),

tissue-culture plastic where cells are ex-

growth factors and biochemicals.

poly(glycerol sebacate) etc) biomaterials

posed to soluble factors in liquid media.

It is clear from this previous list that bio-

can be used. Recently, we have prepared

This is different in the body where the ECM

material design for stem cell applications

a hyaluronic acid-based gel to create a 3D

creates a soft microenvironment where

is progressively abandoning the strategy

microenvironment for the self-renewal of

these molecules are anchored in close

of developing an inert mechanical sup-

human embryonic stem cells [5].

proximity to cell surfaces. This much more

port and adopting the notion that this type

When a greater control over the properties

constrained three-dimensional (3D) niche

of cells need a more dynamic substrate

of the material is required the best option

is a unique microenvironment that has a

capable of directing interactions at the

is to produce synthetic bioactive scaffolds.

prominent role in the maintenance and dif-

cell-material interface and may stimulate

Issues like immunogenicity, pathogen

ferentiation of stem cells. This micro-envi-

and commit cell behaviour through physi-

transmission and purification difficulties

ronment is formed by different components

cal forces, biochemical interactions or to-

have encouraged this option. An example

including cell-cell interactions, extracellu-

pography. This interaction of biomaterials

of a synthetic scaffold is (polyethylene gly-

lar matrix, mechanical properties and se-

with the chemical and physical features of

col) (PEG) gels which can be chemically

creted factors. Collectively, they constitute

stem cells occurs at the micro- and nano

modified to incorporate a compendium of

a complex microenvironment that is diffi-

scales.

bioactive molecules [6]. Immobilization

cult to recapitulate in vitro. Stem cell niche

Cell-cell interaction studies generally rely

seems to increase the stability of the mol-

research uses nanotechnologies to mimic

on co-culture strategies where the effects

ecules, promote persistent signalling and

this microenvironment in order to deter-

of particular molecules are hidden in the

induce receptor clustering [7]. It was re-

mine what are the mechanisms underlying

great complexity of the culturing system.

cently shown that the covalent attachment

the conversion of a stem cell into different

It is therefore difficult to discern the role

of fibroblast growth factor 2 (FGF2) to a

cell types. On the other hand, these biomi-

of soluble or tethered molecules in terms

synthetic nanofibrillar surface composed

metic approaches to create synthetic mi-

of cell-cell interactions. In tissues, the

of a network of polyamide nanofibers re-

croenvironments are very challenging be-

ECM contains many macromolecules such

sulted in the stabilization of the growth

cause there is much we do not understand

as proteoglycans, collagens, laminins,

factor and increased its potency 100-fold

about the natural stem cell niche. Several

fibronectin and sequestered growth fac-

relative to FGF2 in solution. In response to

researchers believe that it may be possible

tors. This molecular repertoire is respon-

the tethered FGF2, embryonic stem cells

to create synthetic stem cell niches that are

sible for the bioactivity of the ECM. For

exhibited increased proliferation through

more bioinspired than biomimetic and po-

example, the sequences of many ECM pro-

activation of mitogenic pathways [8]. An-

tentially more efficient than those observed

teins or receptor ligands are presented to

other example that illustrates the impor-

in nature. Therapeutically, it may be more

stem cells and are recognized by dimeric

tance of ligand presentation in stem-cell

useful to take this bioinspired approach in

cell-surface receptors known as integrins.

fate and function is the immobilization of

the design of the synthetic niche so that it

Binding of integrins to these molecules

leukaemia inhibitory factor (LIF), which led

acts on the stem cells in an unnatural way

can trigger a cascade of signalling events

to more efficient and prolonged activation

to achieve a therapeutic goal [4].

that will impact the gene expression pat-

of LIF targets and maintenance of embry-

Current research efforts in both biomimetic

tern of the stem cell. Therefore, the type of

onic stem cells in an undifferentiated state

and bioinspired strategies are focussing in

ECM molecules that a stem cell encoun-

when compared with soluble LIF [9].

canalBQ_n. 7_DEZEMBRO_2010

39.

ness has a primary role in stem cell line-

factors, and small chemicals present an

age specification. This study reported that

excellent tool to control the differentia-

human mesenchymal stem cells (MSCs)

tion of stem cells. Some of these biomol-

were able to differentiate into tissues that

ecules/chemicals have (i) poor solubil-

had their mechanical properties more

ity, (ii) can be quickly cleaved by cellular

closely mimicked by the polyacrylamide

enzymes, (iii) and have side effects when

A major challenge in tissue engineering is

substrate upon which they were cultured.

administered systemically. Therefore, bio-

to vascularise the transplanted tissue con-

Thus, MSCs that were cultured on rigid

degradable and biocompatible nanoparti-

structs to meet the metabolic demands of

(bonelike) gels differentiated into osteob-

cles able to target stem cells and release

recovery and integration into the organism.

lasts, those that were cultured on medium

the payload in their cytoplasm with conse-

Therapeutic application of the main vas-

stiffness (muscle-like) gels differentiated

quent activation of signalling cascades will

cular signalling molecules (e.g. vascular

into muscle cells, and those that were cul-

be of great interest. Recently, we have re-

endothelial growth factors (VEGFs), FGFs,

tured on more elastic gels (neural-like)

ported the successful delivery of vascular

TGFs, angiopoietins, ephrins and various

differentiated into neural cells [12]. The

growth factors into hESCs, by incorporat-

chemokines) can be a promising approach

acknowledgment that matrix mechanical

ing growth factor-release particles in hu-

to enhance blood supply and neovasculari-

properties impact on stem-cell fate led to

man embryoid bodies (EBs) [15]. These bio-

sation processes around the transplanted

the exploration of further links between

degradable nanoparticles are compatible

tissue. For example, the immobilization of

stem cell behaviour and matrix elasticity.

with cell viability and proliferation and are

VEGF onto a metal substrate using a bio-

Since then, several studies have reported

extremely effective in terms of differentia-

mimetic polymer film was able to promote

that substrate stiffness modulates the

tion. In some cases, the effect on vascu-

the survival and proliferation of endothe-

proliferation and differentiation of em-

lar differentiation of particles containing

lial cells and to induce the differentiation

bryonic stem cells and certain types of

growth factors was superior to the one ob-

of hMSCs into endothelial cells[10].

adult stem cells. For example, adult neu-

served by exposing EBs to large extrinsic

In order to discover novel biomaterials that

ral stem cells cultured on a relatively soft

doses of the same growth factors. Moreo-

have effects on stem cells, high-through-

matrix to mimic brain tissue gave origin to

ver, nanoparticles were taken up by human

put approaches are likely needed. Recent

more neurons than cells grown on a stiffer

embryonic stem cells and accumulated in

efforts have used acrylate-based poly-

synthetic matrix, where glial cells were

the perinuclear region indicating that they

mers spotted in arrays composed of hun-

predominant [13]. Another study found that

could constitute a delivery platform not

dreds of different polymer combinations

the rate of adult skeletal-muscle stem-

only for growth factors but also for other

and found several platforms that could

cell proliferation increased with substrate

type of biomolecules [15].

promote embryonic stem cell attachment,

stiffness [14]. We predict that more stud-

proliferation and differentiation [11]. Simi-

ies will show that the physical properties

Stem Cell Engineering

lar studies must be conducted this time

of culture substrate have a major impact

Various micro-/nanofabrication technolo-

aiming at incorporating many other bio-

on stem-cell fate. With time different cul-

gies have been used to design scaffolds

physical and biochemical parameters in

ture platforms based on soft biomaterials

able to drive the differentiation of stem

this type of high throughput approaches.

are likely to largely replace those made of

cells into specific cell lineages. For ex-

Different matrices, natural and/ or syn-

the standard, rigid, tissue-culture plastic

ample, nanofibers are able to provide an

thetic, can be produced to generate cell-

in order to specifically modulate differen-

in vivo-like extracellular scaffolding to

culture substrates with defined physical

tiation into different fates.

promote regeneration of specific tissues.

characteristics like rigidity (stiffness) and

Usually stem cell cultures are presented

Nanopatterned or nanostructured scaf-

topography. Unlike regular tissue culture

with soluble growth factors and biochemi-

folds are designed to trigger stem cells to

plastic substrates, they provide diffusion of

cals in their culture media. This approach

become specific cell types comprising the

soluble molecules to the basal surface, as

may not always be possible due to specific

tissues and organs in the body.

well as the apical surface. They are espe-

chemical properties of the molecules to

Current research efforts in nanotechnol-

cially interesting in the context of studies

be delivered. Instead, it may be more ben-

ogy applications in tissue engineering are

of homeostatic and disease-related matrix

eficial to deliver these molecules directly

focussing in the following aspects: (i) Mi-

stiffness impact on stem-cell behaviour.

inside the cell to better control their bio-

cro/nano structured scaffolds for tissue

A groundbreaking study by Engler and

availability. Nanoparticles that can carry

engineering; (ii) magnetic nanoparticles

collaborators [12] found that matrix stiff-

molecular payloads of proteins, growth

for magnetic force-based tissue engineer-

40.

canalBQ_n. 7_DEZEMBRO_2010

ing; (iii) nanocomposites for bone tissue

timing and delivery methods, etcIt is of

engineering; and (iv) micro/nanoencapsu-

utmost importance to demonstrate the

lation for cell therapy

long-term safety of these cell-based ther-

The ultimate goal of tissue engineering is

apies. For example, studies in mice have

to recreate the right conditions to support

showed that stem cells injected into the

the massive growth, physical folds and

heart following myocardial infarction gave

twists and cellular and molecular events

origin to mineralized tissue [17]. This was

and ECM derived from the pericardium

of great complexity that occur during re-

possibly due to the reaction of the trans-

of horses can be used as a reconstruc-

generation or replacement of a tissue. The

planted cells to the stiffer mechanical en-

tive material in the dura mater layer of the

general strategy is to grow cells in a scaf-

vironment of the scar tissue that was not

brain meninges following a craniotomy.

fold engineered to define the geometry of

appropriate to induce cardiogenesis.

In a recent development, it was possible

the replacement tissue and provide the

Stem cell based therapy is in hand when

to engineer a bioartificial heart through

right environmental cues that promote tis-

compared with the major challenge that

a decellularization process with deter-

sue regeneration.

is replacing an entire organ with a com-

gents to produce a biocompatible cardiac

Stem cell research has been showing that

plex repertoire of cell types carefully or-

ECM scaffold with a perfusable vascular

stem cells or at least progenitor cells can

ganized to maximize its functional output.

tree, patent valves and a four-chamber-

be isolated from almost every tissue in

Self-organization seems to be an intrinsic

geometry template for biomimetic tissue

the body. With the appropriate conditions

characteristic of cells; cells will cluster

engineering. These researchers man-

it may be possible to stimulate these cells

and communicate with cells that express

aged to populate this ECM scaffold with

to form new tissue. Several studies have

the same cellular adhesion molecules and

an appropriate cell composition, and the

tried to use this biologic intrinsic regener-

under the right conditions can form com-

maturation of this construct developed a

ative potential. Stevens and collaborators

plex structures like the sprouting tubular

nascent pump function [19]. Almost at the

have injected alginate gels or modified hy-

networks formed by endothelial cells lining

same time another group reported the

aluronic acid gels into an artificial space

blood vessels. Simple artificial cell adhe-

transplant of a tissue engineered airway

between the tibia and the periosteum (the

sions have been engineered using biotin

confirming that this approach can in fact

outer lining of the bone). This stimulated

conjugated to cell surfaces and the addition

produce whole-organ tissue engineering

bone and cartilage formation from resi-

of avidin to trigger the assembly of multi-

products that are clinically relevant [20].

dent progenitor cells in the inner layer of

cellular clusters due to the biotin-avidin in-

The scaffold in this case was a decellular-

the periosteum [16]. This is an example of

teraction in order to aid in the development

ized human donor trachea that was seeded

how simple biomaterials can support the

of more complex cellular interactions [18].

with the patients own bone marrow cells

generation of complex tissue by using the

Communication between cells in the tis-

that had been differentiated into cartilage

body as a bioreactor and without the need

sue is essential but a lot of information is

cells. In contrast with traditional trans-

of exogenous cell transplants. In situa-

also coming to cells from their extracel-

plant surgery, the decellularization proc-

tions where the regenerative potential is

lular environment; the scaffold that sur-

ess solved the problem of tissue rejection

low due to different factors like age, trau-

rounds and separates cells within a tissue

because it removed human leukocyte an-

ma, scarring or inflammation like the ones

is a complex material called the Extracel-

tigen traces that are major determinants

that follow myocardial infarction or brain

lular Matrix (ECM). Tissue engineering

in tissue compatibility with the advantage

stroke for example, biomaterial interven-

takes lessons from the characterization

that the patient did not need any immuno-

tions that include cells of external origins

of natural bioactive scaffolds in order to

suppressive drugs [20].

must be included.

construct artificial ones. When possible, a

Both decellularized tissues and synthetic

Several clinical studies with stem cell-

very efficacious strategy is to use cadaver-

scaffolds offer distinct and important

based therapies are currently being per-

or animal-derived decellularized ECM be-

benefits for tissue engineering. Typically,

formed worldwide. Despite the consider-

cause these products have an inherent bi-

biomaterials-engineering approaches fo-

able knowledge gathered in the last years

oactivity to induce regeneration. This type

cus on chemical and/ or physical mecha-

in stem cells biology, further pre-clinical

of approach has found clinical applications

nisms by which the ECM influences cells

and clinical studies are needed to clarify

in routine medical procedures and in life-

and try to reproduce those effectively for a

what is the best stem cell source for cer-

saving scenarios. Products derived from

given tissue. For instance, it may be some-

tain medical applications, the mechanism

the small intestinal submucosa of pigs are

times necessary to work the anisotropic

underlying their regenerative effect, the

used routinely in reconstructive surgery,

features of the culturing system to better

canalBQ_n. 7_DEZEMBRO_2010

41.

teraction of cells with these nanogrooved

electrophysiological application; and (iv)

surfaces was recently analysed by live cell

photothermal nanospectroscopy to identify

imaging [23]. These studies have shown

stem cells in the body.

that cells acquire elongated morphologies

Nanotechnology enables labelling stem

on a surface with nanogrooved patterns and

cells using magnetic, genetic or fluores-

align along that pattern. In this study, the

cent probes which can be monitored by

mimic the tissue. Nanogrooves induced by

dynamic behaviours of living mesenchymal

magnetic resonance imaging (MRI) or fluo-

laser irradiation are an example of this type

stem cells on a nanogroove substrate with

rescence imaging. For example, super-

of approach in bone differentiation studies.

a 200 nm groove depth, an 870 nm ridge

paramagnetic iron oxide (SPIO) nanopar-

The alignment of bone cells and collagen

width and a 670 nm groove width were ob-

ticles can be used to label stem cells and

matrix is closely related to the mechani-

served using time-lapse microscopy. These

analyse their fate in transplantation assays

cal properties of bone. Scaffolds that are

researchers found that filopodia moved

by MRI. In fact, several SPIO nanoparticle

able to promote osteoblast differentiation

as if they were probing the surroundings

formulations (e.g., Feridex/ Endorem and

and modulate their orientation to generate

of the cell protrusion, and then some cell

Ferucarbotran) have FDA (United States

mineralization in a preferred direction are

protrusions invaded the probed areas. Cell

Food and Drug Administration) approval

essential for the generation of biomimetic

protrusions that extended perpendicular to

for human use as MRI contrast agents.

bone tissue. Bangshang Zhu and collabora-

the nanogroove direction tended to retract

The development of nanoparticles for cell

tors, used nanogrooves to induce alignment

more rapidly than those that were parallel

tracking is a multidisciplinary task that

of rabbit mesenchymal stem cell (MSC)-de-

to it. From these observations, the authors

needs highly skilled biological, physical

rived osteoblast-like cells and collagen fi-

hypothesize that the retracting phase of

and chemical expertise. In most cell types,

bres. Nanoscale grooveridge patterns (300

cell protrusions play a role in cell align-

the nanoparticles are taken up through

nm in periodicity, 6070 nm in depth) on the

ment along the nanogroove patterns. Fur-

endocytosis during in vitro cell cultiva-

surface of polystyrene were made by polar-

ther studies using similar live cell imaging

tion and accumulate in the endosomes. Al-

ized laser irradiation. The cells and actin

strategies are required to clearly elucidate

though, some cell types are easier to label

stress fibbers were aligned and elongated

the role of filopodia-mediated cell align-

than others, one has to take into account

along the direction of the nanogrooves. The

ment in these nanopatterned substrates.

the biological features of the cells to be la-

results suggested that nanoscale fibrous

belled and sometimes use chemical tricks

cues in the longitudinal direction might

Stem Cell Tracking and Imaging

to promote the internalization of the nano-

contribute to the aligned formation of bone

To better understand stem cell biology

particles; e.g. mononuclear blood cells are

tissue [21]. A recent study has shown that

and realize the full potential of stem cell

easier to label because by their nature they

osteoblasts are responsive to nanopatterns

therapy, it is essential to monitor the traf-

are primed for internalization of other cells

down to 75 nm in width and 33 nm in depth.

ficking of labelled stem cells by molecular

or molecules by phagocytosis. Also, quite

Nanotexture-driven mineral deposition is

and cellular imaging. Monitorization and

often the internalization of nanoparticles

induced and responsive to even smaller na-

tracking of these cells inside an organism

requires the use of excipients, which may

nopatterns of 50 nm in width and 17 nm in

is a difficult task. This is why stem cells

include peptides and cationic agents [2].

depth. In addition, gene expression of os-

are usually tracked invasively by immuno-

The labelling of stem/ progenitor cells and

teoblast specific markers is upregulated by

histochemistry after removal of tissues or

their transplantation and tracking inside

nanogrooves [22]. These studies indicate

organs from small animals. On the other

the organism may enlighten the dynam-

that nanogrooves can be a very promising

hand, for pre-clinical and clinical trials,

ics of stem cell differentiation, migration

tool to direct the bone response at the in-

it will be fundamental to track stem cells

and therapeutic benefit in several disease

terface between an implant and the bone

noninvasively in order to assess their graft-

scenarios like myocardial infarction, cancer

tissue, which can benefit the installation of

ing and therapeutic effect. Research in this

and neurological conditions. In fact, not so

implants in compromised patients.

area is focussing on the development of the

long ago, Lewis and collaborators succeed-

Although various models have been pro-

following nanotechnologic approaches: (i)

ed in demonstrating that stem/progenitor

posed for how this alignment of cells in

Superparamagnetic iron oxide nanoparti-

cells labelled with magnetic nanoparticles

response to nanopatterns occurs, much

cles for stem cell labelling and diagnostics;

when injected in the blood stream of small

remains to be clarified. Studies with fixed

(ii) quantum dots and fluorophore nanoc-

animals can later be isolated by magnetic

cells do not lend themselves to answering

rystals for stem cell tracking and imaging;

separation after in vivo migration to study

these questions. The dynamics of the in-

(iii) nanoprobes for stem cell detection and

the differentiation of the cells exposed to a

42.

canalBQ_n. 7_DEZEMBRO_2010

biological environment [24]. Although fea-

row emission and broad excitation spec-

sible these type of studies are still limited

trum which allows simultaneous analysis

by technical challenges. In some cases, it

of multiple cell targets by using a single

is difficult to distinguish SPIO-labelled cells

wavelength activation [27]. Qdot conjuga-

from other hypointense regions on MRI im-

tion has been used to follow biomolecules

ages. Such signals can arise from regions

like growth factor receptors, integrins,

containing blood hemoglobin, or blood

phospholipids, and enzymes among oth-

to pluripotent embryonic stem cells. Also,

clots/trombi [25]. The development of new

ers, when stem cells are exposed to differ-

the long-term effects of these nanopar-

nanoparticle formulations based on probes

ent environments or soluble factors [28].

ticles and their degradation products on

other than iron oxide will be of great inter-

In vivo, small animal-tracking of delivered

stem cells should be also assessed at

est for stem cell applications. Some exam-

stem cells has been difficult due to tech-

gene and protein level. Indeed, qdots may

ples have been recently reported based on

nical limitations in terms of labelling but

induce cytotoxic effects due to release of

nanoparticles containing fluorine or man-

also due to the autofluorescent nature of

cadmium triggered by their oxidative deg-

ganese [26].

animal tissues. With imaging platforms

radation [32]. This metal can bind to the

Stem cell differentiation programs are highly

like Calipers IVIS it is now possible to do

sulfhydryl groups of critical mitochondrial

regulated processes that may be sensitive

qdot-tracking in whole animals. Rosen

proteins and induce the production of re-

to nanoparticle internalization. Therefore, it

and collaborators (2007) have reported

active oxygen species, leading to mito-

will be essential to evaluate the long-term

the optimization and validation of a qdot

chondrial dysfunction and ultimately cell

effects of these nanomaterials in the biology

long-term tracking technique of labelled

death [33]. However, it might be possible to

of stem cells. It is possible that the intracel-

mesenchymal stem cells (MSCs) in the

coat qdots in a way that circumvents their

lular degradation of the nanoparticles pro-

mammalian heart. These researchers

in vivo degradation.

duces molecules that are bioactive and have

found that bright qdot crystals were able

potential to activate signalling cascades that

to illuminate MSCs in histological sections

Stem Cell Transfection

can change the differentiation program of

for at least 8 weeks following delivery

Efficient gene delivery systems are re-

the stem cells. The prospect of tracking stem

enabling the complete three-dimensional

quired to fully manipulate stem cell be-

cells with nanoparticle labelling technolo-

reconstruction of the locations of all stem

haviour. This ability is essential for studies

gies is dependent on a careful evaluation of

cells following injection into the heart [29].

of gene function, control of stem cell dif-

their impact on stem cell biology and solving

The use of these nanocrystals for stem

ferentiation, cellular labelling and purifi-

issues like dilution of nanoparticle content

cell-labelling depends on their origin and

cation, and cellular secretion of therapeu-

(and consequent decrease of signal) during

surface modification, mode of internaliza-

tic drugs. Viral methods have been widely

cell division and release by exocytosis. There-

tion and type of stem cells used [30]. Stem

used and have good transduction efficien-

fore, complementary techniques like fluores-

cells are labelled with qdots in several

cies; however they integrate into the ge-

cence must be developed to validate the MRI

ways, including receptor-mediated up-

nome of the host cell. Because of safety

results. Our group is developing nanoparticle

take, lipofection, electroporation, or pas-

issues, non-viral gene delivery systems

formulations that escape the endosome and

sive loading. Under appropriate conditions,

are preferred for stem cell transfection.

combine fluorescent and magnetic labelling

qdots are effective at labelling stem cells

The key challenge in this case is to deliver

to circumvent these issues.

without affecting their self-renewal and

genes to stem cells with high efficiency and

Other nanoparticles that are increas-

differentiation potentials. For example,

low cytotoxicity. Nanotechnology provides

ingly used in cell biology are quantum dots

hMSCs labelled with qdots (0.250 to 16 nM)

invaluable tools for stem cell transfection.

(qdots). These are another class of nano-

maintained their osteogenic differentiation

The main efforts in this area are focuss-

materials usually in the size range of 2-10

potential[30]. Also, intravenous injection of

ing on: (i) Nanomaterials for in vivo gene

nm that can be used for long-term label-

Qdots-labelled mesenchymal stem cells

delivery; (ii) nanowires for gene delivery to

ling of stem cells. Qdots have become a

into NOD/SCID mice (1106 cells) showed

stem cells; and (iii) micro/nanofluidic de-

commercial success because they exhibit

an accumulation after 24 h in the lungs,

vices for stem cell electroporation.

a brighter fluorescent signal, have higher

liver and spleen, but not in the heart, brain

Nanoparticles have been shown to be ef-

photostability (hours) and large stokes

or kidneys [31]. At the moment, most stud-

fective vectors for gene transfection. Green

shift (difference between excitation and

ies were dedicated to labelling multipotent

and collaborators developed a class of pol-

emission wavelengths) than organic dyes

mesenchymal stem cells. Therefore, it

ymers (poly(B-amino esters)) that are able

and fluorescent proteins. They have nar-

will be important to extend these studies

to condense DNA into nanoparticles that

canalBQ_n. 7_DEZEMBRO_2010

43.

These nanodevices are helical structures

a ferromagnetic matrix and is placed in a

of approximately 130 nm in diameter with

MACS Separator. The separator contains a

lengths >100 nm [39], that are able to

strong permanent magnet creating a high-

encapsulate drugs and genetic material.

gradient magnetic field in the magnetisable

These CNTs are internalized by an endocy-

column matrix. Labelled target cells are

tosis independent way and reach the peri-

retained in the column via magnetic force,

facilitate cellular uptake and endosomal

nuclear region after a few hours of contact

whereas unlabeled cells flow through. By

escape. These particles can be coated for

with the cells [40]. After 24 h, a significant

simply rinsing the column with buffer, the

ligand-specific delivery, are biodegradable

number of CNTs have been observed at the

entire untouched cell fraction can be eluted.

and have low toxicity [34]. Another approach

cell nucleus of mesenchymal stem cells

3) Elution of the labelled cell fraction: after re-

used specific recognition of cell surface

[41]. Recent advances on this type of strat-

moving the column from the magnetic field

molecules coupled to an organic-inorganic

egy have produced a novel platform for in-

of the MACS Separator, the retained labelled

hybrid carrier where carbonate apatite na-

tracellular delivery of genetic material and

cells can easily be eluted with buffer.

noparticles were coated electrostatically

nanoparticles, based on vertically aligned

The entire procedure can be performed in

with fibronectin and E-cadherin producing

carbon nanosyringe arrays of controllable

less than 30 min, and both cell fractions,

an efficient gene delivery system for embry-

height. Using this technology, Park and

magnetically labelled and untouched cells,

onic stem cells [35, 36]. These studies with

collaborators have shown that plasmid

are immediately ready for further use, such

nanoparticles reported higher efficiencies

and quantum dots can be efficiently deliv-

as flow cytometry, molecular analysis, cell

for gene delivery and expression than the

ered to the cytoplasm of cancer cells and

culture, transfer into animals, or clinical cell

ones obtained with the leading commer-

human mesenchymal stem cells [42].

therapy applications.

cially available transfection agent, Lipo-

MACS MicroBeads are superparamagnetic

fectamine 2000 [34-36].

Stem Cell Isolation and Sorting

particles made of an iron oxide core and a

Nucleic acids (DNA and RNA) can be deliv-

A key challenge in stem cell research is to

dextran coating. They are nano-sized, rang-

ered in the cytoplasm by the nanoparticles

identify and isolate stem cells from a hetero-

ing between 20 and 150 nm in diameter, and

in a gradual release profile or suddenly,

geneous cell population by a low cost, fast

form colloidal solutions, i.e., they remain

depending if the genetic modulation is in-

and easy procedure. Magnetic or fluorescent

dispersed [43]. Superparamagnetism means

tended to be sustained in time or not. This is

nanoparticles can be used to label stem cells

that in a magnetic field the iron oxide cores

of great advantage when compared with the

followed by magnetic force or flow cytometry

magnetize strongly like ferromagnetic mate-

commercially available options. Indeed, na-

sorting. In the stem cell biology research field

rial, but when removed from the magnetic

noparticles with covalently immobilized DNA

the MACS technology, briefly described

field the particles do not retain any residual

or siRNA were shown to be a very effective

below, is the leading commercial brand and

magnetism. The dextran coating of the Mi-

strategy to regulate gene expression [37,38].

has made the separation of certain stem and

croBeads permits chemical conjugation of bi-

Rosi and collaborators have shown that

progenitor cells a routine procedure.

omolecules. Numerous highly specific mAb,

DNA-gold nanoparticles can have effective

The MACS System is characterized by the

fluorochromes, oligonucleotides and various

intracellular target recognition and binding

use of nano-sized superparamagnetic parti-

other moieties have all been covalently linked

and can be used for antisense gene regula-

cles (approx. 50 nm in diameter), cell sepa-

to MicroBeads, thereby transferring addi-

tion on stem cells [37]. For somatic cells, it

ration columns, and MACS Separators which

tional biochemical and physical properties to

has been reported that these systems have

provide the required strong magnetic field

them [43]. The nano-sized iron-dextran parti-

high resistance to nuclease degradation and

[43]. Magnetic cell separation is performed

cles confer several unique features on MACS

high cellular uptake as a result of their oligo-

in three steps:

Technology. MACS MicroBeads are biode-

nucleotide functionalization. These nanopar-

1) Labelling: cell preparation and labelling

gradable and do not alter cell function. Ef-

ticle systems offer exciting opportunities for

methods are similar to those used in flow cy-

fects on the functional status of cells by mag-

gene expression regulation and the control of

tometry. Each target cell in a cell suspension

netic labelling with MicroBeads are primarily

stem cell fate. Our research group has sev-

is immunomagnetically labelled using MACS

dependent on the target cell surface antigen

eral projects in this area aiming to modulate

MicroBeads, which typically are covalently

and on the degree of crosslinking by mAb or

the differentiation of pluripotent stem cells

conjugated to a monoclonal antibody (mAb)

ligands conjugated to the MicroBeads, but

by the use of nanomaterials.

or to a ligand specific for a certain cell type.

not on the MicroBeads themselves. Cells la-

Other good delivery strategies to transfect

2) Separation: the cell suspension is passed

belled with MicroBeads have been used for

stem cells are carbon nanotubes (CNTs).

through the separation column that contains

numerous functional in vitro assays, experi-

44.

canalBQ_n. 7_DEZEMBRO_2010

mental transfers into animals, and therapeu-

expression of intracellular targets with the

tic transplantations in humans.

fluorescence-quenching beacon [44]. Other

Molecular Detection and Biosensors

and nanoparticles able to quantify enzy-

In addition to detect labelled stem cells, it

matic activities [46]. A recent study reported

is of paramount importance to detect par-

the preparation of polymeric nanoparticles

ticular molecules in the stem cell pathway at

bearing a kinase peptide substrate and

2. Ferreira L, Karp JM, Nobre L & Langer R (2008) New

the cellular level. Nanotechnology provides

near-infrared fluorophore chemically cou-

opportunities: the use of nanotechnologies to manipu-

advanced probes and devices for molecular

pled to the nanoparticle. In the nonphos-

late and track stem cells. Cell Stem Cell 3, 136-146.

detection. For example, (i) carbon nanotube

phorylated state, these nanoparticles have

3. Ferreira L (2009) Nanoparticles as tools to study and

optical probes for single molecule detection

low levels of fluorescence because of the

control stem cells. J Cell Biochem 108, 746-752.

in living cells; (ii) carbon nanotube nanoelec-

short distance between each fluorescence

4. Fisher OZ, Khademhosseini A, Langer R & Peppas NA

trode array for deep brain stimulation; (iii)

probe in the nanoparticle. Upon kinase

(2010) Bioinspired materials for controlling stem cell

nanoparticles for neurochemical detection

phosphorylation of the phosphate groups

fate. Acc Chem Res 43, 419-428.

and biosensors; (iv) nanowires for molecu-

that are incorporated into the peptide sub-

5. Gerecht S, Burdick JA, Ferreira LS, Townsend SA,

lar detection in stem cells; (v) self-assem-

strate the polymeric nanoparticles dissolve

Langer R & Vunjak-Novakovic G (2007) Hyaluronic acid

bly polymeric micelle-based bioassays; (vi)

due to charge unbalance and the fluores-

hydrogel for controlled self-renewal and differentiation

nanoarrays in mass spectrometry for pro-

cence is recovered [46].

of human embryonic stem cells. Proc Natl Acad Sci USA

teomic and metabolomic applications; (vii)

104, 11298-11303.

nanofluidic device for single cell genomic

6. Kraehenbuehl TP, Ferreira LS, Zammaretti P, Hubbell

analysis on a chip.

Conclusion

JA & Langer R (2009) Cell-responsive hydrogel for encap-

The aim of these tools is to monitor biomol-

This report identifies challenges and op-

sulation of vascular cells. Biomaterials 30, 4318-4324.

ecules in real time without using invasive

portunities where nanotechnology can be

7. Irvine DJ, Hue KA, Mayes AM & Griffith LG (2002) Sim-

or endpoint procedures. Currently, most

utilized to advance stem cell research. Al-

ulations of cell-surface integrin binding to nanoscale-

strategies to analyse intracellular bio-

though stem cell nanotechnology is still a

clustered adhesion ligands. Biophys J 82, 120-132.

chemical processes rely on several steps

young discipline, it is already contributing

8. Nur EKA, Ahmed I, Kamal J, Babu AN, Schindler M

of cell-processing like fixation, permeabi-

for new discoveries in stem cell research

& Meiners S (2008) Covalently attached FGF-2 to three-

lization and labelling, which are time con-

and the development of better stem cell

dimensional polyamide nanofibrillar surfaces demon-

suming and expensive when scale-up or

technology. This survey of research topics

strates enhanced biological stability and activity. Mol

high throughput screening is needed. Na-

in stem cell nanotechnology will allow non-

Cell Biochem 309, 157-166.

noparticles can be an appropriate solution

nano-experts to realize the impact that na-

9. Alberti K, Davey RE, Onishi K, George S, Salchert K,

for bio-sensing inside stem cells. Sensors

notechnology is having in both basic stem

Seib FP, Bornhauser M, Pompe T, Nagy A, Werner C &

are usually composed of two parts: one that

cell biology and in translational applica-

Zandstra PW (2008) Functional immobilization of sign-

recognizes and binds the target molecule

tions of stem cell research into medicine.

aling proteins enables control of stem cell fate. Nature

and another that signals the binding event.

Methods 5, 645-650.

One way of doing this is to immobilize the

10. Poh CK, Shi ZL, Lim TY, Neoh KG & Wang W (2010)

recognition molecule to the surface of a na-

Acknowledgements

The effect of VEGF functionalization of titanium on en-

noparticle. This type of approach was used

We acknowledge the support of Crioestaminal, MIT- Por-

dothelial cells in vitro. Biomaterials 31, 1578-1585.

by Hwang and collaborators to monitor

tugal Program, Marie Curie Reintegration Grant, and

11. Anderson DG, Levenberg S & Langer R (2004) Nano-

neuronal differentiation in vivo using a mo-

FCT funding (PTDC/SAU-BEB/098468/2008; PTDC/CTM/

liter-scale synthesis of arrayed biomaterials and appli-

lecular beacon [44]. They have generated a

099659/2008; PTDC/SAU-ENB/113696/2009).

cation to human embryonic stem cells. Nat Biotechnol

quencher-based fluorescent beacon sys-

22, 863-866.

tem to sense the neuron-specific miR124a

12. Engler AJ, Sen S, Sweeney HL & Discher DE (2006)

expression. Moreover this beacon was built

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Protein-phosphorylation-responsive

poly-