Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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20.2.2 Tissue environment and speciﬁc niches

The three-dimensional organization of a tissue is fundamental for the

physiology and behavior of any cell. The local and peculiar structure of a

tissue is called the tissue microenvironment (Spradling et al., 2001). The

spatial architecture around a cell is associated with the interstitial compo-

nents, or extracellular matrix, which consist of structural glycoproteins,

carbohydrates (collagens, elastin, proteoglycans, glycosaminoglycans), and

adhesion compounds (ﬁbronectin, laminin, thrombospondin, etc.), perme-

ated by biological ﬂuids. The cells have a broad range of accessory surface

elements, consisting of the glycocalyx, rich in glycoproteins and glycoli-

pids that interact with extracellular matrix. Finally, intercellular mediators

such as hormones, cytokines, and inﬂammatory and growth factors allow

cellular communication and proliferation control. These mediators interact

with the intercellular matrix, which can generate gradients or hot spots

with high concentrations of molecules that can interact with cell receptors

and provide information on the quality of the tissue and on the necessity

to promote repair or regeneration. Cell differentiation is a result of the

combination of all these elements and interactions. The set of these

molecular interactions characterizes the tissue microenvironment and this

organization gives identity to a tissue. Since an ectopically transferred cell

can modify its physiological activity in response to the new microenviron-

ment, a stem cell of a heterotopic origin differentiates according to the

environmental stimulus (Kai and Spradling, 2003).

Since tissues are spatially ordered by interfaces with solid support

structures, blood vessels or external surfaces, the interactions among

different types of cells determine the behavior of cells introduced during

cell therapy. These interactions control differentiation and facilitate the

formation of functional structures. It is important that a fraction of

transplanted cells keeps the original properties of immature or stem cells,

in order to be able to guarantee the physiological turnover, maintenance of

tissue homeostasis, and natural renewal. In this context, the intercellular

mediators, such as the chemokines, can guide the migration of the

transplanted cell, directing it into an appropriate niche. The molecular

composition of these niches allows some of the stem cells to maintain their

pluripotency, while others respond to the demands for new differentiated

cells. In the bone marrow, this niche is composed mainly of osteoblasts.

The physical contact of these osteoblasts with hematopoietic stem cells is

necessary to support hematopoiesis (Calvi et al., 2003; Zhang et al., 2003;

Balduino et al., 2005; Taichman, 2005).

Once out of the microenvironment, the absence of speciﬁc signals for

the retention of multipotency and self-renewal inevitably induces irrever-

sible and speciﬁc differentiation, leading to the generation of terminally

differentiated cells. Stem cells thus loose their ‘‘stemness’’ and their

primitive or undifferentiated identity (reviewed in Cabrita et al., 2003).

The understanding of ﬁne controls for the maintenance of stemness is the

primary goal for the development of systems that can sustain expansion of

stem cells. Currently, the great challenge is to understand the complex

system involved in the modulation of the intrinsic genetic programs of a

stem cell. Once this information is available, it may be possible by

484 Animal Cell Technology

bioengineering to construct entire fragments of tissues or organs in the

laboratory. These constructs would then be available for implantation into

patients who need repair or regeneration of lost tissues following trauma

or degeneration.

20.3 Applications

Many scientiﬁc groups are now working on cell therapies based on stem

cells. It is difﬁcult to imagine all the possible clinical applications that

could be developed considering the great potential and plasticity of these

cells. Medicine still lacks immediate and efﬁcient solutions for situations

of trauma, tissue dysfunction, or degeneration. Current knowledge is such

that it is possible to produce the necessary cell mass in vitro for therapy,

or to stimulate growth of tissue cells in vivo. It is also possible to construct

in the laboratory, three-dimensional extracellular structures able to pro-

duce biological interactions among cellular elements.

20.3.1 Bioexpansion and biostorage

Finding a way around the shortage of primary material is one of the major

challenges for bioengineering. The most widely studied system is the bone

marrow. Hematopoietic stem cells comprise approximately 0.02% of the

total cells obtained in a good bone marrow aspirate. A similar fraction is

obtained in an umbilical cord blood sample, and a single sample can be

used for transplantation in a 40 kg patient (Cohen and Nagler, 2004).

Current methods proposed for in vitro hematopoietic stem cell expansion

still represent a complex problem, since induction of proliferation is

associated with the loss of stemness. By contrast, skin therapies are

performed with a small healthy biopsy from the patient, since dissociated,

isolated cells can be expanded ex vivo. These expanded cells are useful for

regeneration or fulﬁlling other therapeutic procedures. The same principle

can be applied to mesenchymal cells, which can be expanded in vitro to

regenerate bone tissue.

Currently, several strategies for progenitor cell expansion already exist.

However, the most advanced protocols for hematopoietic cell expansion

still show limited success. Tests with small volumes may work with

conventional culture systems for proliferating blood cells such as lympho-

cytes, in T-ﬂasks under a carbon dioxide (CO

)-rich atmosphere, while

higher volumes are generally used in controlled bioreactors (Noll

et al., 2002; Cabrita et al., 2003). In these systems, ﬁne control is necessary

for all the physicochemical factors, such as pH, temperature, trace ele-

ments, cytokines (Noll et al., 2002). Each cell type requires speciﬁc

conditions.

The key to expanding a stem cell population is to understand the

requirements for proliferation without inducing differentiation. In the case

of self-renewal some elements seem to be essential. Examples are the

homeotic gene HOXB4, the cytokines FLT-3L (fms-related tyrosine

kinase ligand), LIF (leukemia inhibitor factor), interleukin (IL)-3, VEGF

(vascular endothelial growth factor), and proteins of the WNT and

Cell therapies and stem cells 485

NOTCH families, with their ligands (reviewed in Noll et al., 2002;

Cabrita et al., 2003; Sorrentino, 2004).

Once it is possible to expand a clinically deﬁned and safe cell lineage for

transplant, it is also necessary to be able to store the viable cells for

subsequent attempts or procedures. This can avoid further surgery on the

same patient. This demands the development of crypreservation methods

for cells and tissues, involving a tissue bank (blood, skin, bones, cornea,

bone marrow, umbilical cord blood, etc.). Cryopreservation must be

efﬁcient for long periods of storage, since the frozen cells may be required

years after the initial deposit.

20.3.2 Bioengineering

Bioengineering and biomimetics seek to develop tissue scaffolds similar to

the natural ones, in terms of composition and biological behavior. Efforts

are being made to develop products and processes that can substitute tissue

that is biocompatible and allows biointegration.

These objectives are being sought for through distinct models of

reparative cell therapies. For example, hydroxyapatite (HA), a natural

bone constituent, can be synthesized to mold or to ﬁll bone (Marcacci

et al., 1999a, 1999b). The rational strategy for the use of HA is osteocon-

duction and osteoinduction, since it is found in the normal bone tissue and

elicits both adhesion and proliferation of bone cells. This fact also favors

the ﬁnal goal of cell therapy in the bone system, in which the progenitors

are mesenchymal cell that can differentiate into osteoblasts responsible for

the desired ossiﬁcation. In the process of production of HA, it is possible

to create a microporous texture that serves to facilitate the mesenchymal

cell repopulation with autologous bone marrow source. Moreover, this

composition represents an optimum biomimetic system in restorative

therapies. Therefore, it can be produced on an industrial scale, and the

biomaterial interacts well with autologous cell samples.

Another promising example is the use of composite dermo-epidermis

plates whose dermis layer comes from plastic surgery waste or post-

mortem donation. The skin fragment can be acellularized to eliminate the

histo-incompatible cellular components from donor origin. The process

gives an acellular skin structure that can be reconstituted with autologous

epithelial stem cells or autologous keratinocytes in vitro. The ﬁnal dermo-

epidermal graft is appropriate to restore surfaces after severe burns or

removal of tattooing, and to ﬁll cavities in plastic and odontological

surgeries.

A number of other processes are in continuous development. Clinical

applications of these therapies in the urogenital and cardiovascular sys-

tems, in peripheral and central nervous systems, pancreas, joint cartilage

restoration, etc., are being studied. However, almost all procedures suffer

from a common limitation: the availability of donor cells. Cell therapies

have to begin with a relatively high number of cells, and stem cells,

irrespective of their origin, are always a minor subpopulation in tissues.

486 Animal Cell Technology

20.4 Conclusions and perspectives

Reparative medicine and bioprocess engineering are major issues for

current applied biotechnology. Development in these areas is advancing

towards the use of stem cells for therapy. However, the elements and

mechanisms involved in the intrinsic and extrinsic biological control of

these cells are poorly known. All clinical situations performed with auto-

logous transplants of stem cells up to now have generally demonstrated

improvement, or total or partial recovery of tissue function. Reﬁning

techniques in these therapeutic procedures will be necessary to better

characterize and to understand the cellular subpopulations that participate

in restoring the normal physiology of tissues. Concomitantly, engineering

of biomaterials and the associated nanotechnology are appearing as im-

portant tools to speed up this scientiﬁc knowledge. The ultimate goal is to

be able to totally or partially generate an organ with normal physiology, in

a short time period.

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MC, Borojevic R (2005), Bone marrow subendosteal microenvironment harbours

functionally distinct haemosupportive stromal cell populations, Cell Tissue Res.

319:255–266.

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(2003), Hematopoietic stem cells: from the bone to the bioreactor, Trends

Biotechnol. 21:233–240.

Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP,

Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT

(2003), Osteoblastic cells regulate the haematopoietic stem cell niche, Nature

425:841–846.

Cohen Y, Nagler A (2004), Umbilical cord blood transplantation – how, when and for

whom?, Blood Rev. 18:167–179.

Eliaschewitz FG, Aita CA, Genzini T, Noronha IL, Lojudice FH, Labriola L, Krogh

K, Oliveira EM, Silva IC, Mendonca Z, Franco D, Miranda MP, Noda E, de

Castro LA, Andreolli M, Goldberg AC, Sogayar MC (2004), First Brazilian

pancreatic islet transplantation in a patient with type 1 diabetes mellitus, Trans-

plant. Proc. 36:1117–1118.

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potent progenitor cells can be isolated from postnatal murine bone marrow,

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proliferation of ectopic cells, Proc. Natl Acad. Sci. U S A 100:4633–4638.

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P, Quarto R, Martin I, Muraglia A, Cancedda R (1999a), Reconstruction of

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488 Animal Cell Technology

Gene therapy

lio Lopes Silva, Karla de Melo Lima,

Sandra Aparecida dos Santos, and Jose

Maciel Rodrigues Jr

21.1 Introduction

Gene therapy means the introduction of a gene into a cell to correct a

defect. This is intended to slow the progression of a disease and conse-

quently improve an individual’s quality of life. The availability of the

human genome map in 2000 was the ﬁrst major step to enhance the

potential impact of gene therapy as well as cell therapy and tissue

engineering (Romano et al 2000). However, establishing these technologies

requires ex vivo cell cultivation, and one of the challenges is to adapt the

culture from the laboratory to a clinically relevant scale. Thus, the

development of animal cell culture technology has been essential for

advances in gene therapy.

21.2 Gene therapy

With the advent of new knowledge in cell and molecular biology, it has

become evident that the causes of many human diseases are related to

changes in genes. Genes represent the starting point for all events that

subsequently lead to changes in expression of proteins in cells. Protein

function determines, in turn, the cell phenotype and cell function. When

genes are changed in such a way that the proteins encoded by them are

unable to perform their normal function, a genetic disorder may occur.

The cumulative effect of events triggered by atypical or defective genes in

an organ leads to the disease phenotype. Gene therapy is a technique used

to correct genetic defects that may cause the development of diseases, and

consists of blocking an undesirably activated gene, or correcting or

replacing the defective gene with a normal gene (Figure 21.1). The diseases

primarily targeted by human gene therapy are monogenic, that is, those

caused by defects in a single gene (Anderson, 1998). Since the 1990s, the

development of DNA vaccines has led to the concept of gene therapy

being extended to prophylaxis and treatment of infectious diseases (Brun-

nell and Morgan, 1998).

The ﬁrst step in gene therapy is to identify the gene causing the

problem. Then the appropriate genetic material is introduced into the

target cells, with the aim of correcting the problem. Two main strategies

are used: (i) in vivo therapy, in which the genetic material is introduced

directly into the individual by a systemic route, or when possible, at a

speciﬁc site; (ii) ex vivo therapy, in which the target cells are treated with

the gene product outside the individual. The cells are expanded and then

transferred back to the patient (Figure 21.2).

The key to successful in vivo gene therapy is the transfer of the genetic

material to the target cells or tissues and the effective expression of the

gene in question. However, this is a complex process, requiring several

barriers to be overcome. Ex vivo gene therapy involves the transfer of

genes to cells maintained in culture and their subsequent transplantation

to the target tissue. The cells to be transfected must be available in large

amounts, have a long life after transfer to the host, express the gene of

interest for a long time period, and not trigger any kind of immune

response. The advantages of ex vivo therapy include the possibility of

characterizing the modiﬁed cell population before transfer. This makes it

easier to introduce the genetic material into the cells and it can be insured

that the clonal population of cells expresses a high amount of the product

encoded by the transgene before transfer back into the patient.

DNA

mRNA

Protein

DNA

mRNA

Figure 21.1

In a normal cell, genes are transcribed into messenger RNA (mRNA), and

thereafter, translated into proteins with speciﬁc functions (A). When the gene

responsible for encoding a protein has a defect, this may cause the translation of a

defective protein, unable to perform its function and leading to a disease (B). Gene

therapy aims to rectify the defect, by blocking, correcting, or replacing the gene.

490 Animal Cell Technology

21.3 Vectors used in gene therapy

The development of vectors applicable in gene therapy has been the object

of many studies. The vectors that have been developed can be divided into

two categories: synthetic and viral vectors. Synthetic vectors depend on

direct transfer of the genetic information into the target cell, and include

plasmid DNA, naked DNA or DNA associated with some release system.

Some may be applied as DNA vaccines (Ferber, 2001; Niidome and

Huang, 2002). The viral vectors use the capacity of viruses for infection to

introduce the genetic material into the target cells, and are most widely

used in preclinical and clinical protocols for therapies in which a highly

stable transfection is desired. Both systems have advantages and disadvan-

tages, which must be considered depending on the intended therapy

(Cristiano, 1998).

21.3.1 Viral vectors

The concept of viral vectors is based on the innate capacity of viruses to

release their genetic material into infected cells. However, the biggest

concern in using viruses as gene vectors for therapy is that viral infection

may lead to undesired effects such as host tissue destruction. This destruc-

tion may be caused by the induction of genes whose products are harmful

to the host cell, or by the insertion of the viral genetic material into the

host cell genome, leading to pathogeneses, which could include tumor-

Gene

therapy

ex vivo

Gene

therapy

in vivo

Figure 21.2

Strategies applied to gene therapy. Ex vivo – the target cells are treated with the

gene product outside the individual, expanded by cell culture, and then re-

introduced into the patient; or in vivo – the genetic material is introduced directly

into the individual by a systemic route, or, whenever possible, site-speciﬁcally.

Gene therapy 491

igenesis. Other disadvantages include the limitation on the size of genes

that viruses can carry, the difﬁculty of standardizing the production of

viral particles by cell culture, and the higher cost when compared with

plasmid DNA production.

The basis for the use of viruses as gene vectors is the possibility of

separating the components required for replication from those causing

diseases (Hendrie and Russel, 2005). As discussed in Chapters 3 and 18,

some initial information is required:

(i) identiﬁcation of the viral sequence required for replication;

(ii) viral particle construction;

(iii) viral genome packing;

(iv) release of the heterologous gene into the host cell.

Among the viruses widely used for gene therapy are those used to

obtain heterologous proteins from animal cells, particularly RNA viruses

(retrovirus and lentivirus) and DNA viruses (adenovirus, adeno-associated

viruses, and herpesvirus). Table 21.1 summarizes the advantages and

disadvantages of each virus as a carrier in gene therapy.

Adenoviruses are widely distributed in nature, and infect birds and

many mammals, including man. They cause upper respiratory tract infec-

tion, in addition to other infections. Adenoviruses are non-enveloped

viruses with a linear genome composed of double-stranded DNA. Sub-

types 2 or 5 from subgroup C are predominantly used as vectors in gene

therapy. During viral propagation, episomal replication occurs. This

means that the virus genome does not integrate to the host genome, thus

eliminating the risks of mutagenesis due to genomic insertion. The

adenovirus genome is about 35 kb, from which 30 kb may be replaced

with the transgene. Adenoviral vectors are highly effective upon transduc-

tion of target cells, and may be produced in high titre.

Adeno-associated viruses (AVVs) are non-pathogenic human parvo-

viruses. The application of these viruses depends on an auxiliary virus,

usually an adenovirus. AVVs are able to infect dividing or non-dividing

Table 21.1 Advantages and disadvantages of use of different viral vectors in

gene therapy

Viral vectors Advantages Disadvantages

Adenovirus Infection of non-dividing cells Highly immunogenic

Attaining high titre in culture Short-term expression

AVV Infection of non-dividing cells Limited integration

Infection of different cell types Short-term expression

HSV Infection of different cell types Attain low titre in culture

Cytotoxicity

Short-term expression

Lentivirus Attain high titre in culture May cause undesirable effects

Permanent infection of

non-dividing cells

Retrovirus Integration of viral DNA in the

host genome

Infection of dividing cells only

AVV, adeno-associated virus; HSV, herpes simplex virus.

492 Animal Cell Technology

cells and, in the absence of an auxiliary virus, they may integrate into a

speciﬁc site in the host genome. The AVV genome is composed of a

single-stranded DNA molecule. The transgene contained in an AVV may

not exceed 4.7 kb (Zaiss and Muruve, 2005).

Herpes simplex type 1 virus (HSV-1) is a human neurotropic virus, used

as a vector for gene transfer in the nervous system. The HSV genome is

linear, composed of a double-stranded, 152 kb DNA molecule, and able to

contain 40–50 kb of a transgene.

Retroviruses represent a large family of enveloped RNA viruses.

Endogenous retroviruses have been found in all vertebrate species. The

viral particle contains two copies of viral genomic RNA in a conic nucleus.

The viral RNA contains three essential genes, gag, pol, and env. The gag

gene encodes the capsid, matrix, and nucleocapsid proteins, which are

generated by proteolytic cleavage of the precursor protein gag. The pol

gene encodes the viral enzymes: protease, reverse transcriptase, and

integrase. The env gene encodes the envelope glycoproteins that mediate

the virus entry in the cell. After connecting with the receptors on the cell

surface, the capsid containing the genomic RNA enters the cell by fusing

with the membrane. Thereafter, the genomic RNA is converted into

double-stranded DNA by viral reverse transcriptase. The DNA is asso-

ciated with the viral proteins in a complex that is translocated to the

nucleus. An integrase enzyme mediates its integration into the host

genome (Morizono and Chen, 2005). Retroviruses are the most widely

used in gene therapy to date and have the advantage of promoting stable

transfection, because the virus genome is inserted into the host cell

genome. The disadvantages are that this insertion occurs randomly, which

may modify cell genes in a harmful way, and that its particle is fragile,

making handling and attainment of high titre difﬁcult. Additionally, they

infect only dividing cells (Nardi and Ventura, 2004).

Lentiviruses are a subclass of retroviruses expressing a complex that

controls important nuclear functions in the infected cells. Lentiviruses are

able to replicate in non-dividing cells, resulting in infection lasting the

whole life of the host (Quinonez and Sutton, 2002).

21.3.2 Synthetic vectors: plasmid DNA

The advantages of using plasmid DNA as a gene vector are its capacity to

carry a large amount of genetic material, the absence of risk of infection or

mutagenesis, and easy large-scale production from cultivation of bacterial

cells (Brown et al., 2001). The plasmid is a circular DNA, which replicates

independently from the cell chromosome. The plasmid used in gene

therapy should have the following properties:

(i) replication origin in bacteria, to allow efﬁcient replication of plasmids

reaching hundreds of copies per cell;

(ii) a selection gene, which usually provides resistance to an antibiotic, to

allow selection of clones carrying the plasmid;

(iii) a multiple cloning site, which may be cleaved with restriction en-

zymes, allowing insertion of the gene of interest;

Gene therapy 493