**4. Use of stem cells in chronic obstructive pulmonary disease: Experimental basis**

In lungs, affected by chronic inflammation, there is intense production of molecules that signal and can recruit SC (endogenous and transplanted) capable of tissue reconstruction (Rojas *et al*., 2005). In this context, the rationale for cell therapy in COPD comprehends the ability of SC homing toward injured pulmonary tissue, allowing repair of the lung parenchyma and probable clinical efficacy.

Two groups of Japanese researchers reported in 2004 the first consistent results of pulmonary regeneration in an experimental mouse model (C57BL/6 strain) of lesion and later infusion of SC from bone marrow. The mice were submitted to lipopolysaccharide (LPS) intranasal treatment after irradiation. An experimental group received bone marrowderived progenitor cells transplant from transgenic mice donors expressing GFP. There was protection of the lungs against the lesion of the emphysematous type in the animals transplanted with BMMC. It was also detection of stained cells (endothelial and epithelial) only in the recipient animals in which the induced pulmonary lesion (Yamada et al, 2004).

In a model of elastase-induced pulmonary emphysema, Ishizawa et al. (2004) reported that the treatment with retinoic acid or granulocyte colony-stimulating factor (G-CSF) led to the alveolar regeneration and the treatment, concurrently with both factors, resulted in an additive effect. There was BMC mobilization to injured alveoli by retinoic acid and G-CSF besides regeneration process.

Several authors around the world reported experimental and interesting results with cell therapy in animal models of COPD. Some of these works are shortly described in the Table 2.

At our laboratory, several research projects have been directed for the study of morphologic and functional pulmonary recovery after the treatment with ASC in mice with experimentally-induced COPD. Our model basically consists of the induction of emphysema by intranasal instillation of papain or elastase and later treatment with BMMC or MSC pool originated from the bone marrow (Figure 3).

Female mice of the C57BL/6 act as recipients. Transgenic male mice (with C57BL/6 background), which express the green fluorescent protein (GFP) are used as donors of BMMC and MSC for the purpose of cellular tracking and validation of the post transplant chimerism.

The achieved results both in quality and in quantity have shown the regeneration of the pulmonary tissue in animals with emphysema and treated with BMMC pool or MSC (Figure 4).

authors suggest in the same study the importance of paracrine factors derived from MSC as

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

**4. Use of stem cells in chronic obstructive pulmonary disease: Experimental** 

In lungs, affected by chronic inflammation, there is intense production of molecules that signal and can recruit SC (endogenous and transplanted) capable of tissue reconstruction (Rojas *et al*., 2005). In this context, the rationale for cell therapy in COPD comprehends the ability of SC homing toward injured pulmonary tissue, allowing repair of the lung

Two groups of Japanese researchers reported in 2004 the first consistent results of pulmonary regeneration in an experimental mouse model (C57BL/6 strain) of lesion and later infusion of SC from bone marrow. The mice were submitted to lipopolysaccharide (LPS) intranasal treatment after irradiation. An experimental group received bone marrowderived progenitor cells transplant from transgenic mice donors expressing GFP. There was protection of the lungs against the lesion of the emphysematous type in the animals transplanted with BMMC. It was also detection of stained cells (endothelial and epithelial) only in the recipient animals in which the induced pulmonary lesion (Yamada et al, 2004). In a model of elastase-induced pulmonary emphysema, Ishizawa et al. (2004) reported that the treatment with retinoic acid or granulocyte colony-stimulating factor (G-CSF) led to the alveolar regeneration and the treatment, concurrently with both factors, resulted in an additive effect. There was BMC mobilization to injured alveoli by retinoic acid and G-CSF

Several authors around the world reported experimental and interesting results with cell therapy in animal models of COPD. Some of these works are shortly described in the Table 2. At our laboratory, several research projects have been directed for the study of morphologic and functional pulmonary recovery after the treatment with ASC in mice with experimentally-induced COPD. Our model basically consists of the induction of emphysema by intranasal instillation of papain or elastase and later treatment with BMMC

Female mice of the C57BL/6 act as recipients. Transgenic male mice (with C57BL/6 background), which express the green fluorescent protein (GFP) are used as donors of BMMC and MSC for the purpose of cellular tracking and validation of the post transplant

The achieved results both in quality and in quantity have shown the regeneration of the pulmonary tissue in animals with emphysema and treated with BMMC pool or MSC (Figure 4).

the regenerative mechanism operating in the pulmonary parenchyma.

chemotactic mediators by injured organ (Chen et al., 2011).

or MSC pool originated from the bone marrow (Figure 3).

parenchyma and probable clinical efficacy.

besides regeneration process.

chimerism.

**basis** 


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

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

Cell Therapy in Chronic Obstructive Pulmonary Disease: State of the Art and Perspectives 465

Fig. 5. Mean linear intercept (Lm) of the animals in the control and treated groups. N Group - no treatment, EME - instillation of elastase and infusion of DMEM growth medium, ETH - instillation of elastase and infusion of BMMC, E - instillation of elastase only, ETM - instillation of elastase and MSC transplant. Medians followed by the same letter

As it can apprehended from the literature, there is a consistent set of results generated by several laboratories, including those achieved by our research group, which supplied the experimental basis and afforded the cell therapy application by our group in COPD patients.

**5. Clinical application: Cell therapy as a new therapeutic approach for COPD**  Due to the high prevalence and significant economic and social impact caused by COPD, there are, as already presented, several researches in cell therapy, described in animal

The results arising out of the basic research in animal models of COPD cell therapy, at our laboratory, have shown regeneration of the pulmonary parenchyma both in the qualitative and in the quantitative forms, as demonstrated by the histological analyses and by the measurement of the Lm. These results were the grounds for the preparation of a research project submitted to the National Committee of Ethics in Research (CONEP-Brazil) in April 2008. The clinical protocol was approved in April 2009 (registration nº 14764, CONEP 233/2009) and, on May 11th, 2009, the first patient, with CPOD in advanced stage, was

This first work corresponds to a phase 1 clinical screening for the evaluation of safety concerning SC infusion in COPD patients and it was registered with Clinical Trials – NIH – USA (NTC01110252). The experimental outlining consists, basically, of the autologous

models, which sustain the use of ASC in human patients with COPD.

submitted to BMMC pool infusion (Ribeiro-Paes et al., 2011).

indicate no significant difference (p>0,05).

Fig. 4. Pulmonary tissue from female mice C57Bl/6 in representative histological cuts. Hematoxylin and eosin staining. Groups: N - no treatment, E - instillation of elastase only, ETM - instillation of elastase and MSC transplant and ETH - instillation of elastase and infusion of BMMC. Original magnification 200 x.

The regeneration of the pulmonary tissue, expressed in a quantitative way as the measurement of the mean linear intercept (Lm), had a significant statistical difference between animals treated with ASC and controls.

In accordance with the data showed in Figure 5, there is a statistically significant difference between E group, treated with elastase only, and N group, with no treatment, which shows evidence for the efficacy of elastase via intranasal administration in the induction of pulmonary emphysema. Between groups treated only with elastase (E) and treated with elastase and growth medium (EME) there is no statistically significant difference, which suggests the inability of the infusion vehicle in the regeneration of the pulmonary parenchyma. Furthermore, the experimental groups, treated with HSC or MSC have not shown statistically significant difference in comparison with N group, with no treatment. It is worth noting that groups treated with HSC and MSC have not turned out significant difference, which shows the therapeutic equivalence between the two stem strains originated from the bone marrow.

The comparison between the achieved values of Lm equivalent to the groups undergoing the elastase instillation (E) and treated with DMEM (EME), as well as the groups with experimentally-induced emphysema and treated with HSC or MSC has shown statistically significant difference, according to Figure 5 (p>0.05).

Accordingly, it is possible that MSC and BMMC hold a potential role to deliver the required cellular strain diversity during the tissue regeneration process, possibly by paracrine mechanisms (Katsha et al, 2011) and to check, in a significant and effective way, the repair of the lesion in the pulmonary tissue.

Fig. 4. Pulmonary tissue from female mice C57Bl/6 in representative histological cuts. Hematoxylin and eosin staining. Groups: N - no treatment, E - instillation of elastase only, ETM - instillation of elastase and MSC transplant and ETH - instillation of elastase and

The regeneration of the pulmonary tissue, expressed in a quantitative way as the measurement of the mean linear intercept (Lm), had a significant statistical difference

In accordance with the data showed in Figure 5, there is a statistically significant difference between E group, treated with elastase only, and N group, with no treatment, which shows evidence for the efficacy of elastase via intranasal administration in the induction of pulmonary emphysema. Between groups treated only with elastase (E) and treated with elastase and growth medium (EME) there is no statistically significant difference, which suggests the inability of the infusion vehicle in the regeneration of the pulmonary parenchyma. Furthermore, the experimental groups, treated with HSC or MSC have not shown statistically significant difference in comparison with N group, with no treatment. It is worth noting that groups treated with HSC and MSC have not turned out significant difference, which shows the therapeutic equivalence between the two stem strains

The comparison between the achieved values of Lm equivalent to the groups undergoing the elastase instillation (E) and treated with DMEM (EME), as well as the groups with experimentally-induced emphysema and treated with HSC or MSC has shown statistically

Accordingly, it is possible that MSC and BMMC hold a potential role to deliver the required cellular strain diversity during the tissue regeneration process, possibly by paracrine mechanisms (Katsha et al, 2011) and to check, in a significant and effective way, the repair of

infusion of BMMC. Original magnification 200 x.

between animals treated with ASC and controls.

significant difference, according to Figure 5 (p>0.05).

originated from the bone marrow.

the lesion in the pulmonary tissue.

Fig. 5. Mean linear intercept (Lm) of the animals in the control and treated groups. N Group - no treatment, EME - instillation of elastase and infusion of DMEM growth medium, ETH - instillation of elastase and infusion of BMMC, E - instillation of elastase only, ETM - instillation of elastase and MSC transplant. Medians followed by the same letter indicate no significant difference (p>0,05).

As it can apprehended from the literature, there is a consistent set of results generated by several laboratories, including those achieved by our research group, which supplied the experimental basis and afforded the cell therapy application by our group in COPD patients.
