Methods Were recruited from the pulmonology ambulatory 20 grade 3 COPD (GOLD 2017) in a 12-month follow-up (Figure 1) and study was approved by the Institutional Review Board. Inclusion aged between 40 to 70 years undergoing optimized treatment for COPD with grade 2 or 3 that is, they were all using beta-2 long-action agonists associated with long-acting anticholinergic and inhaled corticosteroids; a forced expiratory volume in the first second (FEV1) of 30-50%, they have quitted tobacco use for at least six months and could be submitted to the pulmonary rehabilitation program. Exclusion: absence of emphysema on chest tomography; infection or history of infectious disease for less than 3 months; previous history of coronary artery disease; presence of pulmonary hypertension; use of home oxygen therapy; advanced hepatic or renal insufficiency; detection of immunosuppressive or infectious diseases; and presence of known neoplasms.

1. Autologous steam cells infusion (ASCI) in COPD patients: impact on
quality of life and on physical performance
Introduction
Chronic obstructive pulmonary disease (COPD) is considered a serious public health
disease because millions of people are worldwide affected. COPD is an irreversible but
preventable and treatable illness characterized by persistent respiratory symptoms and
airflow limitations due to disruption of the airways and alveolus chronically exposed to
noxious particles and gases [1,2].
Different clinical approaches for pulmonary rehabilitation undoubtedly have contributed
to life prolongation and QL improvement of COPD patients. To evaluate the variable
individual responses, however, physical functional tests and subjective questionnaires
should be used to complement the dynamic objective clinical assessment. Among them,
the Six-Minute Walk Test (6MWT) [3] and the Saint George Hospital Respiratory
Disease Questionnary (SGRQ), are the most used tools to monitor the treatment
effectiveness and to establish clinical prognosis [4]. New and advanced procedures have
been postulated but established curative guidelines are lacking yet. Actually the
optimized therapeutic in course are meant to reduce the symptoms and to improve the
QL. Cell therapy with stem cells arises as a promising alternative procedure [1,5-11].
There are growing evidences (and hope) of its high potential to regenerate damaged
tissues and to correct dysfunction of organs primarily affected by any pathological
process [5, 8, 10,12-14].
Several cell types have been investigated for ASCI-therapy, among which the bone
marrow mononuclear cells (BMMC) and the mesenchymal derived stem cells (MSCs)
[5, 11, 15-16]. Preliminarily reports refer to their effectiveness in cellular therapy of
1

2. pulmonary diseases [5,8,10,12-14]. Yet, there is no consensus of the effective stem cells
migration and organ regeneration [14].
Koc et al. (2000) [17], reported that a double ASCI associating MCS-AT and BMMC,
produced a rapid hematopoietic response, without side effects, in advanced stage breast
cancer carriers submitted to intense chemotherapy [17,18]. No equivalent study is
known with COPD patients.
The goal was to evaluate the impact of ASCI with MCS-AT and BMMC, separately or
co-infused, exploring the complementary parameters used to evaluate the QL and the
(PP) of patients with intense COPD.
Methods
Were recruited from the pulmonology ambulatory 20 grade 3 COPD (GOLD 2017) in a
12-month follow-up (Figure 1) and study was approved by the Institutional Review
Board. Inclusion aged between 40 to 70 years undergoing optimized treatment for
COPD with grade 2 or 3 that is, they were all using beta-2 long-action agonists
associated with long-acting anticholinergic and inhaled corticosteroids; a forced
expiratory volume in the first second (FEV1) of 30-50%, they have quitted tobacco use
for at least six months and could be submitted to the pulmonary rehabilitation program.
Exclusion: absence of emphysema on chest tomography; infection or history of
infectious disease for less than 3 months; previous history of coronary artery disease;
presence of pulmonary hypertension; use of home oxygen therapy; advanced hepatic or
renal insufficiency; detection of immunosuppressive or infectious diseases; and
presence of known neoplasms.
2

3. The patients were divided in four groups of five, randomly distributed using numbered
and sealed envelopes that made it impossible to visualize their contents. The groups
were defined as follows:
1) Control: No intervention was performed but for the usual drug treatment according to
GOLD 2017[1]. And were observed over a 12 months.
2) BMMC: received by surface arm vein an infusion of 1x 108
BMMC obtained after
puncture of the posterior iliac crest and gradient differential centrifugation; for the
infusion the BMMC were taken in a 30-ml of physiological saline.
3) MCS-AT: were infused with 1x 108
MCS in a 30-ml suspension of physiological
saline. The cells were obtained by a adipose tissue by abdominal liposuction, removing
about 150 ml of fat (tissue and infused fluid). This was cultured in 75 cm3
bottles for 21
days until the culture had a 2-3 passage and were infused into a peripheral vein at the
established described above;
4) Co-infusion group (CoINF): received a mix of MCS-AT + BMMC co-infusion of
stem cells as described above. MCS-AT were collected 40 days before BMMC and
cultive; the collection, isolation, and culture of BMMC were performed on the day of
co-infusion.
All the procedures and infusions were done in the Hospital, and were monitored.
Clinical evaluation during follow-up: immediately after the cell infusion, on day 7 and
at 1, 3, 6, 9, and 12 months afterwards.
Three major criteria were considered: 1) Physical performance: All participants done
the 6MWT to evaluate their PP was carried out in a 21 meters and at a walking pace as
fast as possible for the patient. The oxygen saturation (SatO2) and the heart rate (HR)
3

4. were monitored with a non-Onyx [19] portable pulse oximeter. The exertion related to
dyspnea and fatigue of lower limbs were evaluated by the modified Borg scale [20].
The patients progress on PP was determined by the grade changes (Δ) in results (6MWT
+ scores) at each follow-up visit minus the grade obtained on the preceding evaluation.
2) QL questionnaire: At every visit were asked to complete a QL questionnaire (SGRQ)
translated and validated in Brazil by Sousa et al. [21]. This questionnaire provides score
to evaluate three domains: 2.1) symptoms, 2.2) physical activity, and 2.3) the
psychosocial impact of the respiratory disease, aiming at better detection of clinical
improvements [22]. The responses were evaluated for the total questionnaire and for
each of its domain considering that a 4% reduction or a 10% reduction in each domain
between visits, was a significant improvement. The patient progression in terms of QL
was calculated relatively to the preceding evaluation score, as described above for the
6MWT.
3) Clinical status and Exacerbation: the third and more important criterion to evaluate
the patients´ medical improvement during follow-up.
Statistical analysis
This was a phase II preliminary study, so no sample calculation was performed.
An initial descriptive analysis of the results was performed. The qualitative
characteristics were described by scores and presented as median and 25-75% of the
score range limits. Data was also shown by values relative to their basal ones before
treatment. Quantitative characteristics were described by medians and their respective
confidence intervals (95% CI). When applicable Stata 11.0 (Stata Corp., College
Station, TX, USA) was used for the analyses. The data obtained after application of the
4

5. SGRQ were analyzed using either the overall score or the scores at each questionnaire
domain.
Results
The flow chart at Figure 1 summarizes the present study. The patient selection and the
group formation in Figure 2.
As shown in Table I, the experimental groups were all homogeneous according to the
basal parameters: the patients aged 56 to 67 years, women prevailing; the same disease
severity (FEV1 30 ≤ 50%); the average Body Mass Index (BMI) was 24-26 kg/m2
.
Before treatment the PP measurements at 6MWT, the HR and the SatO2 did not differ
among groups; QL evaluated by the total SGRQ scores had a baseline of 29-38
points(Table-I).
No side effects were observed after infusion of stem cells and no patiead yo be
excluded during the experiment. Therefore, the Stem Cell (SC) therapy was considered
safe for all engaged patients.
Follow-up: The QL scores derived from the SGRQ application increased in the Control
group throughout the 12 months follow-up indicating a considerable clinical worsening
of the COPD status (Figure 3). Relatively to the basal values (100%), the QL-scores in
the control group raised somewhat irregular but persistently to 46.7% above the baseline
score at the end of the follow-up period.
The QL-scores in the SC-treated groups decreased relatively to the respective basal
values indicating a persistent improvement of the patients´ well-being. The Control
group QL-scores was high at every corresponding time of re-evaluation. At 12 months
5

6. of follow-up the decreased QL-scores were 47.6% (Co-INF), 34.2% ( MCS-AT) and
10.4% (BMMC) less. (Table 2; Figure 3).
Analysis of the symptom´s domain taken from the whole QL questionnaire (SGRQ)
confirmed that the groups treated with mesenchymal stem cells (SCM-AT and Co-INF
groups) improved symptoms after 3 months treatment. This effect was not evident for
the BMMC group. The scores of the QL-symptoms´ domain relatively to the basal
values are in Figure 3. The Co-INF group had a symptoms improvement steady through
the 6- and 9-month visits; a significant increase of 10% over baseline at the 12-month
visit. In the disease-impact domain of SGRQ the same pattern was observed in all
groups, but for the 12-month visit, the CoINF group showed a more significant
improvement Figure 3.
Individual signs and symptoms indicative of COPD exacerbation were eventually seen
in SC-treated groups but they were more intense and frequent in the Control group
which had always some patients with exacerbation to be taken care. At the 9th
month
return, for example, two (2) patients with exacerbation signs were evaluated in the
Control group, 2 in the BMMC, 1 in the MCS-AT and 3 in the Co-INF group.
Apparently the increased scores recorded in SMT-AT and CoINF groups at the 9 month
re-evaluation (Figure 3-4) coincided with these exacerbation cases which obscured the
long term positive follow-up effects. These are better seen in the SGRQ symptoms
domain of Figure 4; for the 6MWT, the distances covered by the patients at any
follow-up session did not differ from the preceding runs after considering the general
criteria for significance (Δ>50 meters) [22], (Table 3),
The heart rate (HR) measured 1-min after the forced walk increased less than
relatively to the initial HR. The change was greater at the CoINF Group (Δ=-11bpm).
6

7. The final HR compared to the 2-min HR result (considered the effort) did not change,
indicating a low cardiac demand in a period of significant effort. (Table 4)
Similarly, the Borg scale for dyspnea and limb fatigue did not significantly change,
indicating that the forced walk did not require great effort, although it was sufficient to
increase the HR.
Finally no change between the measurements of SatO2 done 1 min after the forced
walk at rest before run, or at 2-min of the recovery after run time The maximum
decrease in SatO2 in the SC treated groups was less than 2% throughout the 12-month
follow-up. (Table 5).
The results did not show significant changes in the PP parameters used to calculate the
SGRQ scores.
Therefore, if PP does not seem to be altered and exacerbation crises eventually occur in
every group, the mechanism(s) responsible for the improved QL referred by the patients
after treatment remains subjective and unclear; perhaps not have been observed by the
small sample.
Discussion
COPD is a serious disease with irreversible lung disruption that does not revert with the
regular medicines used to relieve cough and dyspnea. Its clinical setting is progressive
and exacerbation is usual throughout life. According to Silva (2011) COPD patients
may adapt to the disease and may perceive the illness impact only when the frequency
or intensity of symptoms increase [23]. Therefore, the QL of COPD patients should
correlate with both the disease duration and with the frequency of the exacerbation
crises.
7

8. The present results have shown a positive change in the QL of all COPD patients treated
with autologous stem cell infusion. The QL improvement was clear in patients treated
with MCS-AT and in those CoINF with MCS-AT + BMMC, but it was less intense in
the group treated with BMMC alone. Further, in the two former groups improvement
was evident from the third month after the cell infusion to the follow-up end.
Contrasting these results, the data indicated that the Control group presented a
progressively worsening disease despite the controlled use of optimized COPD
therapy.,
Physical parameters used to evaluate QL-scores did not change in any group. The
QL-improvement indicated by the SGRQ scores was apparently due to a decrease in
two domains extracted from the subjective questionnaire: the disease impact and the
symptoms domains.
Exacerbations were common in every group. However, at 9 months, there was a small
worsening, which can be explained by the number of events (exacerbations) reported in
the visits by each group , and may also be related to the timing of the disease.
Ribeiro-Paes et al (2011) [24] reported the results of BMMC autologous infusion to
COPD patients, an improvement of the QL was reported by the treated patients but not
those in the control group, suggesting that SC can alter the QL. However, no assessment
instrument was used in that study; it is noteworthy that the treatment group had a lower
number of exacerbations at one year of follow-up when compared to the control group ,
suggesting that SCs may interact positively; that is, patients may have increased QL
because they have a lower number of exacerbations.
The results have shown also, that measurements during the 6MWT, did not change
during follow-up, i.e., there were no significant difference in the distance covered ,
8

9. either on the Borg scale or as measured by SatO2 however HR was increased
suggesting that the forced walking test demanded some effort from the patients.
However, the amount of effort did not require extra work and energy because O2
desaturation was observed. Some authors [25,26] did not observe correlations between
the improvement in the QL and the distance covered in the 6MWT, but they reported
that the improvement of the endurance time was related to the distance covered in the
6MWT. In addition, they found correlations with the walk test improvements, impact
domain and the total score of the SGRQ at follow-up, which corroborates with our
findings.
According to Solway [27], the 6MWT is presented as a complementary tool in the
evaluation of physical endurance, in monitoring the treatment effectiveness and as a
way to establish the prognosis of individuals with COPD. Wise [28] showed that a gain
of 54 m in the 6MWT after intervention showed a correlation with clinical
improvement. Wise [28] showed clinical significance with a difference of 50 to 80 m in
the 6MWT. Therefore, the best indicator of gain is related to the functional
improvement of the patient, with a gain equal to or greater than 10% of the distance in
the baseline 6MWT. In the present study, we did not observe a significant change
(difference greater than 50 m) in the distance covered between the evaluated periods,
but there was no deterioration of the physical capacity of the patients studied.
According to Puente [29], a decrease in SatO2 ≥ 4% suggests an important desaturation
during exercise. The maximum decrease in SatO2 of the treated groups was less than
2%; that is. In the CoINF group, saturation increased by 1.5% in the 12-month period,
which may be positive for this group of patients, since it leads to an increase in exercise
tolerance and, consequently, an improvement in PP.
9

10. One limitation of this study was that the parameters of QL could be influenced by their
subjective nature. Patients from the treated groups probably had a high expectation of
improvement in terms of dyspnea and, therefore, the subjectivity of the patients may
have influenced the results. Nevertheless, the treated groups had superior results to the
control group, including the number of exacerbations. Another limitation that should be
emphasized was the small number of patients in each group. Further studies should,
therefore, be performed with a larger number of patients in order to evaluate the
reproducibility of the results and to consolidate the efficacy of SC therapy in COPD.
Conclusions
The results suggested that SC treatments, especially autologous MCS-AT and the
combination of autologous MCS-AT + BMMC (CoINF) tended to positively modify QL
scores without deteriorating PP after 12 months of follow-up, when compared
conventional treatment. The analysis of other parameters of this study support these
findings and increase the knowledge about the potential of MCS-AT and BMMC and
their combined use. Thus, cellular therapy offers a new therapeutic approach with great
potential for the treatment of COPD.
References
[1] GOLD. Global Initiative for Chronic Obstructive Lung Disease: global strategy for
the diagnosis, management, and prevention of chronic obstructive pulmonary disease
(Revised, 2017). 2011; Available at: http://www.goldcopd.org
[2] Diretrizes Brasileiras para o manejo da DPOC (Adaptação para o Brasil do
Consenso Latino-americano de DPOC). Last updated on 23/08/2016. Documento
Oficial da Sociedade Brasileira de Pneumologia e Tisiologia Website. Available at:
http://www.sbpt.org.br.
10

11. [3] ATS Statement: Guidelines for the six-minute walk test. Am J Respir Crit Care Med
2002;166:111-7.
[4] Rodrigues,LR. Avaliação do programa pulmonar. In: Rodrigues, L. R. Reabilitação
Pulmonar: conceitos básicos. São Paulo: Manole, 2003.
[5] Ribeiro-Paes JT, Bilaqui A, Greco OT, Ruiz MA, Alves-deMoraes LBC, Faria CA,
et al. Terapia celular em doenc¸as pulmonares: existem perspectivas? Rev Bras Hematol
Hemoter. 2009;31:140---8.
[6] Hind M, Maden M. Is a regenerative approach viable for the treatment of COPD? Br
J Pharmacol 2011;163:106-15.
[7] Rennard SI, Wachenfeldt K. Rationale and emerging approaches for Targeting lung
repair and regeneration in the treatment of chronic obstructive pulmonary disease. Proc
Am Thorac Soc 2011;8:368-75.
[8] Ribeiro-Paes JT, Bilaqui A, Greco OT, Ruiz MA, Marcelino MY, Stessuk T, Faria
C.A, Lago MR. Unicentric study of cell therapy in chronic obstructive pulmonary
disease / pulmonary emphysema. Int J Chron Obstruct Pulmon Dis 2011;6:63-72.
[9] Faria CA, las Heras RK, Stessuk T, Ribeiro-Paes JT. Experimental Basis and New
Insights for Cell Therapy in Chronic Obstructive Pulmonary Disease. Stem Cell Rev
2012;8:1236-44.
[10] Longhini-dos-Santos N, Barbosa-Oliveira VA, Kozma RH, Faria CA, Stessuk T,
Frei F, Ribeiro-Paes JT. Cell Therapy with Bone Marrow Mononuclear Cells in
Elastase-Induced Pulmonary Emphysema. 2012. Stem Cell Rev 2012;1-9.
[11] Ribeiro-Paes JT, Stessuk T, Kozma RdlH. Cell therapy in chronic obstructive
pulmonary disease: state of the art and perspectives. In: Kian-Chungong, editor. Chronic
obstructive pulmonary disease --- current concepts and practice. Rijeka: InTech; 2012.
p. 455-74.
11

12. [12] Zhen G, Liu H, Gu N, Zhang H, Xu Y, Zhang Z. Mesenchymal stem cells
transplantation protects against rat pulmonary emphysema. Front Biosci
2008;1:3415-22.
[13] Abreu SC, Antunes MA, Pelosi P, Morales MM, Rocco PR. Mechanisms of
cellular therapy in respiratory diseases. Intensive Care Med 2011;37:1421-31.
[14] Wada H, Yoshida S, Suzuki H, Sakairi Y, Mizobuchi T, Komura D, Sato Y, Yokoi
S,Yoshino I. Transplantation of alveolar type II cells stimulates lung regeneration during
compensatory lung growth in adult rats. J Thorac Cardiovasc Surg 2012;143:711-9.
[15] Iyer SS, Co C, Rojas M. Mesenchymal stem cells and inflammatory lung
diseases.Panminerva Med 2009;51:5-16.
[16] Prockop DJ, Oh JY. Mesenchymal stem/stromal cells (MSCs): role as guardians of
inflammation. Mol Ther 2012;20:14-20.
[17] Koc ON, Gerson SL, Cooper BW et al. Rapid hematopoietic recovery after
coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal
stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin
Oncol 2000;18:307–16.
[18] Lee ST, Jang JH, Cheong J W et al. Treatment of high-risk acute
myelogenousleukaemia by myeloablative chemoradiotherapy followed by co-infusion
of T cell-depleted haematopoietic stem cells and culture-expanded marrow
mesenchymal stem cells from a related donor with one fully mismatched human
leucocyte antigen haplotype. Br J Haematol 2002;118:1128-31.
[19] Sciurba F, Criner GJ, Lee SM, Mohsenifar Z, Shade D, Slivka W, et al. Six-minute
walk distance in chronic obstructive pulmonary disease: reproducibility and effect of
walking course layout and length. Am J Respir Crit Care Med 2003;167:1522-7.
12

13. [20] Borg GA. Psychophysical bases of perceived exertion. Med Sci Sports Exerc
1982;14:377-81.
[21] Sousa TC, Jardim JR, Jones P. Validação do Questionário do Hospital Saint George
na Doença Respiratória (SGRQ) em pacientes portadores de doença pulmonar
obstrutiva crônica no Brasil. J Pneumol 2000;26:119-28.
[22] Bonilha, AF, Chiodi, ST, Gonçalves, LO, Panizzi, EA & KerKoski, E. (2007)
Mensuração da qualidade de vida em pessoas com doença pulmonar obstrutiva crônica
através do Saint George s Respiratory Questionnaire (SGRQ). In: XI Encontro Latino
Americano de Iniciação Científica e VII Encontro Latino Americano de Pós-Graduação,
2007, São José dos Campos - SP. Programação e Anais do XI Encontro Latino
Americano de Pós-Graduação. São José dos Campos - SP: Universidade do Vale do
Paraíba, v. 1, pp. 1193- 1196
[23] Silva MS. Qualidade de vida relacionada à saúde de pacientes com Doença
Pulmonar Obstrutiva Crônica [dissertation]. São Paulo: Universidade de São Paulo;
2011.
[24] Ribeiro-Paes JT, Bilaqui A, Greco OT, Ruiz MA, Marcelino MY, Stessuk T, et al.
Unicentric study of cell therapy in chronic obstructive pulmonary disease/pulmonary
emphysema. Int JChron Obstruct Pulmon Dis 2011;6,63-71.
[25] Laviolette L, Bourbeau J, Bernard S, Lacasse Y, Pepin V, Breton MJ, et al.
Assessing the impact of pulmonar rehabilitationon functional status in COPD. Thorax
2008;63:115-21.
[26] Dourado VZ, Antunes LCO, Tanni SE, Godoy Fatores associados à diferença
clinicamente significativa da qualidade de vida relacionada à saúde após
condicionamento físico em pacientes com DPOC J Bras Pneumol 2009;35:846-53.
13

14. [27] Solway S, Brooks D, Lacasse Y, Thomas A. A qualitative systematic overview of
the measurement properties of functional walk tests used in the cardiorespiratory
domain. Chest 2001;119:256-70.
[28] Wise RA, Brown CD. Minimal clinically important differences in the six-minute
walk test and the incremental shuttle walking test. COPD 2005;2:125-9.
[29] Puente ML, García PJ. Lung function tests in clinical decision-making. Arch
Bronconeumol 2012;48:161-9. http://dx.doi.org/10.1016/j. arbr.2011.12.007
14