Application of Nanotechnology in Stem Cell and Cell Reprogramming

Introduction

Stem cells are the parent cells of all tissues and organs of the body and exist mainly to

maintain and replace the cells in the areas where they are found such as blood, bone

marrow, skin, muscle and organs like the brain, liver, etc. Embryonic stem cells (ESCs)

and adult stem cells are the two types of stem cells. Embryonic stem (ES) cells are the cells

of the inner cell mass of a blastocyst, an early-stage embryo. ES cells are pluripotent and

give rise during development to all three primary germ layers: ectoderm, endoderm and

mesoderm. It means that they can develop into more than 200 cell types of the adult body.

As pluripotent ES cells are capable of developing into any cell type, they can be used to

study emerging cell transplantation therapies which inturn aids in understanding a wide

array of diseases and conditions, from diabetes and Alzheimer’s disease to cancer and

spinal cord injuries. Adult stem cells (somatic stem cells), are stem cells which maintain

and repair the tissue in which they are found. They can be found in children, as well as

adults.

Cellular Reprogramming is the conversion of one specific cell type to another.

Specifically, direct reprogramming is the conversion of a somatic cell type, such as a

fibroblast, to a pluripotent cell type known as an induced pluripotent stem cell, or iPS cell.

The development of reprogramming technologies and delivery methods are shown in

figure 1.

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Nanotechnoly deals with the design, construction, and utilization of functional structures

which are measured in 1-100 nanometres. Materials and systems because of the small size

can be used to improve physical, chemical, and biological properties, phenomena and

processes.

Stem cells and nanotechnology are two different fields which are highly interdisciplinary.

By combining these two fields, a new emerging field is formed, that is, stem cell

nanotechnology, which refers to the application of nanotechnology in stem cells research

and development. The application of nanomaterials and nanotechnology in stem cells

research and development provides a new chance to solve current problems related to stem

cells research and development. For example, magnetic nanoparticles (MNPs) have been

successfully used to isolate and sort stem cells, quantum dots have been used for molecular

imaging and tracing of stem cells, nanomaterials such as carbon nanotubes (CNTs),

fluorescent CNTs have been used to deliver gene or drugs into stem cells. Unique

nanostructures were designed for controllable regulation of proliferation and differentiation

of stem cells, and all these advances speed up the development of stem cells toward the

application in regenerative medicine.

In particular, the effects of structure and properties of nanomaterials on the proliferation,

differentiation, and reprogramming of stem cells have become significant for regeneration

medicine and material science. Following are some applications of nanotechnology in stem

cell and cell reprogramming.

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Figure 1: Reprogramming Timeline

Application of Nanotechnology in Stem Cells

Application of Magnetic Nanoparticles in Isolation of Stem Cells

Magnetic nanoparticles have superparamagnetic property and they have been widely used

in applications such as hyperthermia, magnetic resonance imaging (MRI), tissue repair,

immunoassay, drug/gene delivery, cell separation, etc. Different studies have shown that

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MNPs can directly label stem cells, following the labeled stem cells isolation by magnetic

force or flow cytometry. MNPs combined with Cd34 antibody, successfully enriched

peripheral blood progenitor cells (PBPCs) from human blood circulation.

By using quadrupole magnetic flow sorter (QMS), CD34+ cell immunomagnetic labeling

and isolation from fresh leukocyte fraction of peripheral blood was done. These isolated

CD34 progenitor cells can be used for patient therapy. Cells isolated by using magnetic

nanoparticles are listed in table 1.

Table 1: Cells isolated with magnetic nanoparticles.

Cell type Cell marker Reference

Mesenchymal stem cells CD117+ (De Coppi et al., 2007)

Human hematopoietic stem cell CD133+ (Suzuki et al., 2006)

Endothelial cells CD34+ (Wang et al., 2007)

Human embryonic stem cells SSEA-4 (Fong et al., 2009)

Application of Nanotechnology in Stem Cell Therapy

Nanotechnology plays significant role in stem cell therapy. A research has shown that the

combination of nanoparticles with adult stem cells results in atherosclerotic plaque

destruction and also restoring of artries. Silica-gold nanoparticles were infused into the

heart of pigs along with adult stem cells. Arterial plaque burnt away after laser light

heating of nanoparticles. In another study it was demonstrated that walking function was

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recovered after injecting nanofiber conjugated with the laminin and nerve stem cell, in

paralyzed mouse with spinal cord injury. Neuraxis extension can be stimulated by the

neurite sprouting in combination with the integrin, which adjust cell differentiation

stimulate. As a result nerve stem cells begin to differentiate on damage position and

generate new neuron which inhibit colloid cell form cicatrix and help recovery nerve.

In several animal models neointimal hyperplasia after arterial interventions can be

inhibited by using nitric oxide (NO). Nitric oxide in combination with nanofiber delivery

vehicles can create a novel, more potent NO-releasing therapy that can be used clinically.

Optimizing the Stem Cell Environment

In stem cell research a significant area of study is the extracellular environment and how

differentiation, migration, adhesion and other activities are controlled by the conditions

outside the cell through signaling. The extracellular matrix (ECM), provide direction for

stem cell activities with the aid of molecules such as collagen, elastin, and proteogylcan.

Different nanoparticles have been engineered that mimic the ECM, for studying their

effects on stem cells.

Nanoscale scaffolds improve cell survival by aiding the engrafting process. Channels for

alignment of stem and progenitor cells are provided by nanofibers spun from synthetic

polymers such as poly lactic acid (PLA), or natural polymers of collagen, silk protein or

chitosan.

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Nucleotide Delivery for Genetic Control

Genetic controls, using DNA or siRNA, can be used as a tool for cellular functions control

in stem cells, particularly for directing their differentiation. Traditionally used viral vectors

such as retroviruses, that can cause complications in whole organisms such as inducing

mutations leading to cancer, can be replaced by using nanoparicles.

Nanoparticles offer vectors which are less expensive, easily producible for transfection of

stem cells, and also have low risk of immunogenicity, mutagenicity or toxicity. Cationic

polymers interaction with DNA and RNA molecules is a popular approach. Different smart

polymers can be developed, with features such as targeted delivery or scheduled release.

Drug and nucleic acid delivery into mammalian cells has been tested by using carbon

nanotubes with different functional groups. it was reported in a study that the mixing of

nanopticles formed of carbonate apatite crystals with cell adhesiproteins fibronectin and E-

cadherin-Fc results in the enhancement of transgene delivery and also accelerates

expression in recipient mouse embryonic stem cells (mESCs) compared to carbonate

apatite alone. These mixed organic and inorganic nanoparticle composites bind to the cell

surface and become internalized by mESCs, providing an efficient nonviral method of

gene delivery. In another study it was demonstrated that higher efficiency transfection of

human embryonic stem cells (hESCs) can be achieved through the development and use

biodegradable nanoparticles of poly (β-amino esters) that incorporate plasmid DNA within

the nanoparticle. Transfected hESCs reportedly maintained their viability, undifferentiated

state, and pluripotency following transfection with nanoparticles.

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Carbon nanotubes in combination with DNA or peptide molecules have high potential in

gene or peptide storage, and delivery system in molecular therapy of diseases. Table 2

enlists some other nanotechnological approaches used as delivery system.

Table 2: List of delivery systems

Delivery system Delivery of Delivered to Reference

Exosomes siRNA Mesenchymal Alvarez et al., 2011

stem cells

Liposomes siRNA Human CD34+ Martino et al., 2009

HSCs

PLGA SOX9 Human MSCs Jeon et al., 2012

Gene

Nanoneedles Green Fluroscent Human MSCs Han et al., 2008

Protein (gene)

Plasmonic GFP Human T cells Hleb et al., 2010

plasmid
nanobubbles

Nanorobot Antibodies Natural killer cells Dougla et al., 2012

Apatite nanoparticle E-cadherin Embryonic stem cells Kutsuzawa et al.,

2006

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Stem Cell Tracking and Imaging

Molecular and cellular imaging is done for the trafficking of labeled stem cells to

understand the stem cell biology and realize the full potential of stem cell therapy. Stem

cell labeling is enabled by nanotechnology by using magnetic, genetic or fluorescent

probes which can be monitored by magnetic resonance imaging (MRI) or fluorescence

imaging. A unique insight into cell migration can be offered by labeling embryonic stem

cells (ESCs) with nanoparticles prior to transplantation into animal models. Furthermore,

nanoparticle tracking may also contribute to replace lost cells or to the repair of the

diseased state. Thus, scientists were able to track mouse embryonic stem cells (mESC)

migration to a site of transient cerebral ischemic lesion by transfecting mESCs with

superparamagnetic dextran-coated iron oxide nanoparticles followed by transplantation

into rat brains. By using magnetic resonance imaging (MRI), it was observed that the

morphology of migrated mESCs had altered to neural-like cells in contrast to the rounded

morphology of mESCs at the implantation site. In a study, it was reported that the use of a

superparamagnetic iron oxide agent to label mESCs prior to injection into a murine model

of acute myocardial infarction has showen a reduction in thinning of the anterior and

anterolateral region of the myocardium and partial restoration of left ventricular volume

end fraction.

Quantum dots have been used to successfully label mouse embryonic stem cells (mESCs),

with minimal effects on viability and proliferation, and to visualize these cells within mice

following transplantation. Different nanoarticles have been used for labeling stem cells,

some of them are enlisted in table 3.

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Table 3: Summary of different studies performed with nanoparticles used to label stem

cells

Nanoarticle Stem Cell Type Labeling Approach Reference

QDs MSC Fluorescence Chen et al. 2015 
imaging

QDs Neural stem and Fluorescence Slotkin et al. 2007 

progenitor cells imaging

SPIO Central nervous Magnetic resonance Guzman et al. 2007 

system cell imaging

SPIO (Ferumoxides) MSC Magnetic resonance Amsalem et al.,

imaging 2007 

Silica shell on SPIO MSC Magnetic resonance Kim et al. 2010 

(ferumoxides) and fluorescence
imaging

Silica-coated gold MSC Photoacoustic Jokerst et

imaging al. 2012 

Gold MSC Photoacoustic Nam et al. 2012 

imaging

Application of Nanoparticles in Stem Cell Tissue Engineering

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Stem cell based therapeutic strategy to human diseases is developed by the combination of

stem cells with engineering principles. Stem cells are guided to develop into three-

dimensional tissue constructs by various nanofabrication technologies. For example,

nanofibers are able to provide in vivo-like extracellular scaffolding to promote

regeneration of specific tissues. Stem cells are triggered by the designed nanopatterened or

nanostructured scaffolds to become specific cell types compromising the tissues and

organs in the body.

Nanofabrication technologies have been used to guide stem cells to develop into 3D

biodegradable scaffolds. Stem cells can be triggered to become specific cell types

compromising the tissues and organs in the body, by use of nanostructured scaffolds. Cells

deposit their own matrix inside these scaffolds and as the scaffold degrades, they form a

3D tissue structure that mimics the body’s natural tissues. For example, in a study it was

reported that a 3D cell culture system has been developed by using a designer peptide

nanofiber scaffold with mouse adult neural stem cells. 18 different peptides were

synthesized which trigger functional motifs to promote cell adhesion, differentiation, and

bone marrow homing activities.

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Application of Nanotechnology in Cell Reprogramming

Use of Nanotechnology Based Vectors in Cell Reprogramming

Efficiency of induced pluripotent stem cells (iPSC) reprogramming can be improved by

the use of nanoparticle-mediated gene transfection. The delivery system of nanoparticle-

based gene carriers has been thought to be potentially safer than viral-mediated delivery.

Moreover, these polymer complexes can protect DNA from nuclease degradation. In

addition, these nanoparticles are small enough to enter the cell by endocytosis. Because of

their biocompatibility and physiochemical properties, polyurethanes (PUs) are used as

biomaterials in tissue engineering and in hydrogels. Cationic polyurethane are the

nanoparticles having a higher transfection efficiency with lower cytotoxicity. In a study it

was demonstrated that Polyamidoamine dendrimer-modified magnetic nanoparticles have

ability to induce the reprogramming of human dermal fibroblasts to pluripotent cells.

Furthermore, it was reported that efficient pluripotency in mouse fibroblasts was induced

to produce transient expression of four embryonic stem cell (ESC) transcription factors by

using biodegradable cationic polymer PEI-coated superparamagnetic nanoparticles.

Carbon-based nanomaterials can be used as efficient vector because of their ability to

attach various molecules, including cell-specific ligands, to carbon molecules. This ability

allows them transport genes to targeted sites such as transcription factors, oligonucleotides,

small RNAs, synthetic molecules, or chemicals, so that, to regulate gene expression. So

these carbon nanomaterials can be used for the generation of reprogrammed cell types as a

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virus-free alternative to existing methods. Some of the nanotechnology based delivery

system used in cell reprogramming, are summarized in table 4.

Table 4: Nanoparticulated Delivery System-Based Applications on iPSC Research

Carrier/Platfor Transfection Cells for Significant Reference

Factors Reprogramming Results
m
Liposomal Plasmid, pCX Mouse iPSCs Park et al., 2012
magnetofection -OKS-2 A, and embryonic generated via
(LMF) pCX-cMyc fibroblasts LMF obtained
(MEFs) pluripotency in
vitro and in
vivo
PLGA/bPEI Viral Mouse Compared to Seo et al., 2013
nanoparticles constructs embryonic liposome–DNA
containing fibroblasts complex,
Oct4 and Sox2 (MEFs) PLGA/bPEI
nanoparticles
showed higher
capacity of
DNA carrying
Polyketal Embryonic Mouse bone Induced Sohn et al., 2013
nanoparticles stem cell- marrowderived pluripotent-
specific hematopoietic gene and
microRNAs cells protein
expression in
(BM-MNCs) cultured
BM-MNCs
Cationic Recombinant Human foreskin A stable cargo– Khan et al., 2013
bolaamphiphile protein: Sox2, fibroblasts protein
Nanog, Klf4, complex is
and NR5A2 created using
cationic
bolaamphiphile
Poly-b-amino Human Polycistronic Higher Montserrat
esters fibroblasts plasmid transfection et al., 2011
efficiency was
achieved

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NanoScript for Cellular Reprogramming

Researchers have developed an artificial, nanoparticle-based transcription factor (TF),

termed NanoScript, which is designed to mimic the structure and function of TFs. It is a

nanoparticle-based artificial transcription factor for Effective Gene Regulation. It was

constructed by combining functional peptides and small molecules called synthetic

transcription factors, onto gold nanoparticles.

NanoScript effectively transcribes targeted genes on endogenous DNA in a non-viral

manner. It has significance in cellular reprogramming and stem cell differentiation and was

uesd to target the MyoD gene, responsible for inducing muscle differentiation, resulting in

differentiation of muscle cells. Furthermore, pluripotent cells or neurons can also be

generatedby targeting pluripotent genes or neural-specific genes with nanoscript.

Use of Nanogrooved Substrate in Direct Lineage Reprogramming of Fibroblasts

It was demonstrated in a study that the direct conversion of somatic fibroblasts into

induced dopaminergic (iDA) neurons could be effectively enhanced by nanotopographical

stimulation. For example, when the mouse embryonic fibroblasts (MEFs) were cultured

with microgrooved substrates, direct reprogramming of MEFs to iDA neurons was

induced. Analysis with quantitative reverse transcription polymerase chain reaction (qRT-

PCR) has revealed that nanogrooved substrates increased the expression of DA marker

genes including neuronspecific class III beta-tubulin (Tuj1), vesicular monoamine

transporter 2 (VMAT2), tyrosine hydroxylase (TH), dopamine transporter (DAT), and

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aromatic L-amino acid decarboxylase (AADC). Based on these results it was demonstrated

that the strongest influence can be exerted by nanogrooved substrate on the direct lineage

reprogramming of MEFs to iDA neurons. Therefore, these results suggest that the direct

lineage reprogramming to iDA neurons can be effieciently done by use of nanopatterned

subtrates, and its effectiveness ensures that substrate nanotopography plays a significant

role in the cell fate changes during direct lineage reprogramming.

Use of Graphene Based Substrates to Enhance Cell Reprogramming

Graphene and its derivatives are considered to be ideal platforms for supporting cell

growth and differentiation. It was reported that graphene promotes the reprogramming of

mouse somatic cells into induced pluripotent stem cells (iPSCs). To check the efficiency of

graphene, mouse embryonic fibroblasts (MEFs) were plated on the graphene substrate. It

was observed that a significant increase in colonies undergoing reprogramming was

exhibited by graphene substrate cultures. Higher numbers of positive cells in the graphene-

coated substrate cultures were shown by using alkaline phosphatase (AP) staining. The

reprogramming efficiency was quantified by performing fluorescence-activated cell sorting

(FACS) analysis for Oct4-GFP-positive iPS cells derived from Oct4-GFP KI MEFs in

graphene-coated substrate. Moreover, higher levels of pluripotency marker genes were

shown in graphene-coated substrate cultures, by quantitative RT-PCR analysis. So, form

these results it has been suggested that graphene can be used to enhance the efficency of

the reprogramming.

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