**3. Stem cells and cell therapy: The rationale for use in the lung**

The employment of cells for treating diseases is an ancient therapeutic practice, which dates back to the transfusion of whole blood or platelet concentrate in different acute or chronic clinical conditions. The first hematopoietic stem cells (HSC) transplantations were made according to the works of Till and Mculloch in 1961, on the response of mice with the bone marrow transplanted after lesion by ionizing radiation. Since then, new ranges and possibilities of use of other tissues according to the experimental model adopted by the authors have arisen.

The potential of differentiation of stem cells (SC), i.e., the wide range of options of commitments available for the cell (Smith, 2006), has aroused a growing and great interest, bearing in view the employment in the therapy of several types of degenerative diseases and in tissue bioengineering (Atala, 2008). According to The National Institutes of Health (NIH), SC can be defined as cells able to divide for indefinite time *in vitro* and to give origin to specialized cells. Melton and Cowan (2004) proposed a working definition of SC: "a clonal self-renewable entity which is multipotent and can generate several types of differentiated cells." Notwithstanding the concept variation, SC have two basic characteristic aspects: selfrenewal, in order to maintain the pool of undifferentiated cells for tissue replacement, remodeling, and repair, as well as the differentiation into at least one mature cell type. These inherent properties for SC are afforded through particular asymmetric divisions, where undifferentiated cells are originated, or, alternatively, differentiation into specialized cells (Figure 2).

Fig. 2. Assimetric SC division. An undifferentiated SC under microenvironment stimulation start assimetric divisions producing two distinct daughter cells. One cell, undifferentiated, maintain the SC pool. In contrast, the differentiated cell acquires a new mature and specialized phenotype.

Cell Therapy in Chronic Obstructive Pulmonary Disease: State of the Art and Perspectives 461

the lungs, arising out of the radiation, provided high levels of incorporation in the alveolar

In the same year, Kotton *et al*. (2001) IV infused Lac-Z stained cells of transgenic mice into recipient wild animals, which underwent pulmonary lesion by intratracheal instillation of bleomycin. There was typical staining of lac-Z expression (Incubation in medium containing X-gal), with statistically significant increase in the animals sustaining lesion with bleomycin. The grafted cells showed evidence for morphologic and molecular phenotype of pneumocytes type I. So, cultured or fresh aspirates of bone marrow cells can express pulmonary markers. Thereby, these cells could represent a potential therapy in extensive

An elegant experimental model of suppression of bone marrow and later lesion with bleomycin was elaborated by Rojas *et al*., (2005). The authors obtained full survival index and protective effect in mice which underwent MSC transplantation. The immunohistochemistry analysis of the pulmonary tissue of the animals with suppression of bone marrow disclosed, when compared to group without suppression, that the transplanted cells (GFP+) were present in the organ and in a large number, even 14 days

As in the animal models, cell migration and chimerism were also observed in human patients who received, for different reasons, bone marrow allogeneic transplant, as in the models of animal studies. Suratt et al. (2003), in a pioneer work, showed pulmonary chimerism upon the incorporation of cells with Y chromosome in women receiving HSC allogeneic transplant from male donors. Another study, 7 patients who underwent pulmonary transplant between (donor and recipient) individuals of opposite sexes showed, by means of different assays of histochemistry staining and molecular analysis (RT-PCR), the presence of mesenchymal stem cells (MSC) in lungs of recipients with cytogenetic expression of the sex of donor. In a period of up to 11 and a half years after the transplant

Nevertheless, the SC migration to the lungs can be overestimated and, therefore, they are allegedly present at a much lower rate with a questionable clinical meaning. So, the results obtained and reported have been evaluated more carefully by some authors, who challenge the accuracy of the employed detection techniques. For example, after transplanting MSC GFP+ in mice which had previously received an LPS intraperitoneal injection, Xu et al. (2008) did not find, in the immunohistochemistry analysis of the pulmonary tissue conducted 14 days after the transplant, circumstantial evidence for a significant presence of cells with positive sign of GFP. However, although the authors did not find evidence for an actual integration of MSC to the pulmonary tissue and the presence of cells with the pulmonary phenotype, there was demonstration that the SC transplant afforded a decrease in the lungs inflammation and edema induced by the LPS. There are, accordingly these results, the indication that the action mechanism of cells would be mediated by paracrine factors that stimulate tissue regeneration rather than cell

More recently, Katsha and collaborators (2011) reported a significant improvement resulting from the use of MSC from the murine bone marrow for the repair and regeneration of the pulmonary parenchyma, in an elastase-induced experimental model of emphysema. The

was verified donor cells in the recipient patients (Lama et al., 2007).

tissue.

alveolar degeneration.

after the administration of bleomycin.

engraftment into lungs (Huh et al., 2011).

Considering their origin, SC are classified in three general types: embryonic stem cells (ESC), germinative stem cells (GSC), and adult or tissue-specific stem cells (ASC). The ESC are derived from the inner cell mass of the blastocyst, capable to generate any differentiated cellular type of the three primary germ layers (ectoderm, mesoderm and endoderm), as well as the GSC originated from the gonadal crest (Geijsen et al., 2004). On the other hand, ASC are undifferentiated cells, found in differentiated cell types in a tissue where they can renew themselves for long periods of time, and can differentiate to yield specialized cell types of the host tissue. By and large, ESC maintains the undifferentiated stage for a long period of time without losing their differentiation potential (Draper et al., 2004). Moreover, the ASC have a limited number of generations, and at each division there is loss of response to differentiation signals (Jiang et al., 2002).

The knowledge that undifferentiated cells exist in the bone marrow has been verified since the 40's decade; by the way, the blood progenitors are the first well characterized SC. Both in humans and in animal models, the literature reports consistent data with evidence for the existence of stained SC from the bone marrow in lungs after bone marrow transplant (Bittmann et al ., 2001; Kotton et al., 2001; Krause et al., 2001; Lama et al., 2007; Ribeiro\_Paes et al., 2009; Schrepfer et al., 2007; Suratt et al., 2003; Yamada et al., 2004). At different experimental situations, these and others classical works have shown evidence for the migration of SC to the lung and have provided the theoretical reference which gives grounds for the idea of employing cell therapy in the regeneration of pulmonary tissue.

The experimental evidence of migration of SC from the bone marrow to the lungs was pioneering described in the work of Pereira et al. in 1995. Authors cultured murine cells expressing a collagen human gene and injected the expanded mesenchymal precursor cells into irradiated mice. The presence of transplanted cells in recipient animals for a period of up to 5 months was showed by PCR *in* situ assay. There was incorporation into the pulmonary tissue, where the cells disseminated through the mesenchymal parenchyma and could continue the replication process *in* vivo. Therefore, bone marrow cells can migrate and populate the pulmonary tissue and act as precursors of local cells.

Experimental animal models and clinical trials in regenerative tissue therapy by intravenous (IV) SC or BMMC infusion indicate a "pulmonary first-pass effect" as proposed by Fischer et al. 2009. The lungs act as a barrier, where administered cells are preferentially attracted and retained. Cell size and adhesion receptors of the stem and progenitors cells IV infused can determine this effect through pulmonary microvastulature (Fischer et al., 2009). Five minutes after labeled MSC IV infusion was verified, in animal model, a significant greater bioluminicensce signal in the lungs, in relation to several other organs, such as heart, spleen, liver and kidney. Therefore, the mean size of injected cells larger than the caliber of lung capillaries provides an efficient and fast cell trapping in lungs (Schrepfer et al., 2007).

Interesting works found on literature indicate the initial migration and chimerism in lungs after cell transplantation. Krause et al. (2001) transplanted male mice cells into females with bone marrow depleted by ionizing radiation and tracked the presence of Y chromosome in gastrointestinal tract, liver, lung and skin. It was verified co-staining of pneumocytes type II and Y chromosome in bronchi and alveoli showed by FISH assay (Y chromosome and surfactant B mRNA staining) and immunohistochemistry (anti-cytokeratin antibodies for the detection of epithelial cells). However, authors proposed that the significant damage to

Considering their origin, SC are classified in three general types: embryonic stem cells (ESC), germinative stem cells (GSC), and adult or tissue-specific stem cells (ASC). The ESC are derived from the inner cell mass of the blastocyst, capable to generate any differentiated cellular type of the three primary germ layers (ectoderm, mesoderm and endoderm), as well as the GSC originated from the gonadal crest (Geijsen et al., 2004). On the other hand, ASC are undifferentiated cells, found in differentiated cell types in a tissue where they can renew themselves for long periods of time, and can differentiate to yield specialized cell types of the host tissue. By and large, ESC maintains the undifferentiated stage for a long period of time without losing their differentiation potential (Draper et al., 2004). Moreover, the ASC have a limited number of generations, and at each division there is loss of response to

The knowledge that undifferentiated cells exist in the bone marrow has been verified since the 40's decade; by the way, the blood progenitors are the first well characterized SC. Both in humans and in animal models, the literature reports consistent data with evidence for the existence of stained SC from the bone marrow in lungs after bone marrow transplant (Bittmann et al ., 2001; Kotton et al., 2001; Krause et al., 2001; Lama et al., 2007; Ribeiro\_Paes et al., 2009; Schrepfer et al., 2007; Suratt et al., 2003; Yamada et al., 2004). At different experimental situations, these and others classical works have shown evidence for the migration of SC to the lung and have provided the theoretical reference which gives grounds for the idea of employing cell therapy in the regeneration of

The experimental evidence of migration of SC from the bone marrow to the lungs was pioneering described in the work of Pereira et al. in 1995. Authors cultured murine cells expressing a collagen human gene and injected the expanded mesenchymal precursor cells into irradiated mice. The presence of transplanted cells in recipient animals for a period of up to 5 months was showed by PCR *in* situ assay. There was incorporation into the pulmonary tissue, where the cells disseminated through the mesenchymal parenchyma and could continue the replication process *in* vivo. Therefore, bone marrow cells can migrate and

Experimental animal models and clinical trials in regenerative tissue therapy by intravenous (IV) SC or BMMC infusion indicate a "pulmonary first-pass effect" as proposed by Fischer et al. 2009. The lungs act as a barrier, where administered cells are preferentially attracted and retained. Cell size and adhesion receptors of the stem and progenitors cells IV infused can determine this effect through pulmonary microvastulature (Fischer et al., 2009). Five minutes after labeled MSC IV infusion was verified, in animal model, a significant greater bioluminicensce signal in the lungs, in relation to several other organs, such as heart, spleen, liver and kidney. Therefore, the mean size of injected cells larger than the caliber of lung

capillaries provides an efficient and fast cell trapping in lungs (Schrepfer et al., 2007).

Interesting works found on literature indicate the initial migration and chimerism in lungs after cell transplantation. Krause et al. (2001) transplanted male mice cells into females with bone marrow depleted by ionizing radiation and tracked the presence of Y chromosome in gastrointestinal tract, liver, lung and skin. It was verified co-staining of pneumocytes type II and Y chromosome in bronchi and alveoli showed by FISH assay (Y chromosome and surfactant B mRNA staining) and immunohistochemistry (anti-cytokeratin antibodies for the detection of epithelial cells). However, authors proposed that the significant damage to

populate the pulmonary tissue and act as precursors of local cells.

differentiation signals (Jiang et al., 2002).

pulmonary tissue.

the lungs, arising out of the radiation, provided high levels of incorporation in the alveolar tissue.

In the same year, Kotton *et al*. (2001) IV infused Lac-Z stained cells of transgenic mice into recipient wild animals, which underwent pulmonary lesion by intratracheal instillation of bleomycin. There was typical staining of lac-Z expression (Incubation in medium containing X-gal), with statistically significant increase in the animals sustaining lesion with bleomycin. The grafted cells showed evidence for morphologic and molecular phenotype of pneumocytes type I. So, cultured or fresh aspirates of bone marrow cells can express pulmonary markers. Thereby, these cells could represent a potential therapy in extensive alveolar degeneration.

An elegant experimental model of suppression of bone marrow and later lesion with bleomycin was elaborated by Rojas *et al*., (2005). The authors obtained full survival index and protective effect in mice which underwent MSC transplantation. The immunohistochemistry analysis of the pulmonary tissue of the animals with suppression of bone marrow disclosed, when compared to group without suppression, that the transplanted cells (GFP+) were present in the organ and in a large number, even 14 days after the administration of bleomycin.

As in the animal models, cell migration and chimerism were also observed in human patients who received, for different reasons, bone marrow allogeneic transplant, as in the models of animal studies. Suratt et al. (2003), in a pioneer work, showed pulmonary chimerism upon the incorporation of cells with Y chromosome in women receiving HSC allogeneic transplant from male donors. Another study, 7 patients who underwent pulmonary transplant between (donor and recipient) individuals of opposite sexes showed, by means of different assays of histochemistry staining and molecular analysis (RT-PCR), the presence of mesenchymal stem cells (MSC) in lungs of recipients with cytogenetic expression of the sex of donor. In a period of up to 11 and a half years after the transplant was verified donor cells in the recipient patients (Lama et al., 2007).

Nevertheless, the SC migration to the lungs can be overestimated and, therefore, they are allegedly present at a much lower rate with a questionable clinical meaning. So, the results obtained and reported have been evaluated more carefully by some authors, who challenge the accuracy of the employed detection techniques. For example, after transplanting MSC GFP+ in mice which had previously received an LPS intraperitoneal injection, Xu et al. (2008) did not find, in the immunohistochemistry analysis of the pulmonary tissue conducted 14 days after the transplant, circumstantial evidence for a significant presence of cells with positive sign of GFP. However, although the authors did not find evidence for an actual integration of MSC to the pulmonary tissue and the presence of cells with the pulmonary phenotype, there was demonstration that the SC transplant afforded a decrease in the lungs inflammation and edema induced by the LPS. There are, accordingly these results, the indication that the action mechanism of cells would be mediated by paracrine factors that stimulate tissue regeneration rather than cell engraftment into lungs (Huh et al., 2011).

More recently, Katsha and collaborators (2011) reported a significant improvement resulting from the use of MSC from the murine bone marrow for the repair and regeneration of the pulmonary parenchyma, in an elastase-induced experimental model of emphysema. The

Cell Therapy in Chronic Obstructive Pulmonary Disease: State of the Art and Perspectives 463

function, decreases airspace enlargement

Improves alveolar parameters (mean alveoli area and linear interval)

Attenuates cigarette induced emphysema, restores the increased Lm, increase pulmonary microvastulature,

lung perfusion, cellularity and ECM content.

Decreases inflammation and airspace enlargement, prevents cigaretteinduced weight loss, restores cigaretteinduced BM dysfunction

Ameliorates alveolar structure, restores increased Lm and destructive index

Therapeutic effects Probable action

mechanism

Inhibition of the apoptosis of alveolar cell wall

> Paracrine effects

> Paracrine effects

> Paracrine effects

> Paracrine factors

Reference

al., 2006

Liu et al., 2008

Huh et al., 2011

Ingenito et al., 2011

Schweitzer et al., 2011

Katsha et al., 2011


Animal COPD induction Stem cell type /

Rat Papain

Rat Cigarette smoke for 6 months

Mice Cigarette smoke for 6 months

Mice Elastase MSC /

Co-60

source

MSC / bone marrow

BMC MSC Conditioned medium of MSC/bone marrow

Sheep Elastase MSC/ lung Increases tissue mass,

Human or murine MSC / cell-free contidioned medium adipose tissue

bone marrow

Fig. 3. Experimental design of protease-induced emphysema and ASC treatment.

Table 2. Experimental animal models of cell therapy for COPD.

Rabbit Elastase BMMC Improves pulmonary

authors suggest in the same study the importance of paracrine factors derived from MSC as the regenerative mechanism operating in the pulmonary parenchyma.

Notwithstanding the diversity of used methodologies, in human patients and animal models, has been proposed that ASC from several tissue sources can migrate and populate injured areas in the lung. It is propounded that the regenerative property of SC involves cellular migration to the site of tissue damage and probable promotion of functional and structural organ repair. This mobilization process (homing) is related to liberation of chemotactic mediators by injured organ (Chen et al., 2011).
