Vol.2, No.4, 155-162 (2012) Stem Cell Discovery
http://dx.doi.org/10.4236/scd.2012.24020
Long-term effect of autologous progenitor cell
therapy to induce neo angiogenesis in patients with
critical limb ischemia transplantated via
intramuscular vs combined intramuscular and distal
retrograde intra venous
Luis Padilla1,2*, Juan Rodriguez-Trejo3, Ignacio Escotto3, Manuel López_Hernandez4,
Mauricio González5, José De Diego6, Neftaly Rodrgiuez3, Jesús Tapia2, Takeshi Landero1,
Carranza Pilar Hazel1, Horacio Juarez Olguin1, Mauricio Di Silvio1,7, Paul Mondragon-Teran8
1Department of Experimental Surgery, Microsurgery Unit, Centro Medico Nacional “20 de Noviembre” ISSSTE, Mexico City,
Mexico; *Corresponding Author:
2Surgery Department, Faculty of Medicine, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico
3Angiology, Vascular and Endovascular Surgery Unit, Centro Medico Nacional “20 de Noviembre” ISSSTE, Mexico City, Mexico
4Haematology Unit, Centro Medico Nacional “20 de Noviembre” ISSSTE, Mexico City, Mexico
5Bank of Blood Unit, Centro Medico Nacional “20 de Noviembre” ISSSTE, Mexico City, Mexico
6Pediatric Hematology Unit Centro Medico Nacional ‘20 de Noviembre’ ISSSTE, Mexico City, Mexico
7Department of Research, Hospital General de México OD, Secretaria de Salud México, Mexico City, Mexico
8Department of Biomedical Research, Centro Medico Nacional “20 de Noviembre” ISSSTE, Mexico City, Mexico
Received 13 August 2012; revised 15 September 2012; accepted 11 October 2012
ABSTRACT
Critical limb ischemia is a medical condition that
decreases blood flow and limb oxygen supply;
this disease in its late stages of progression
leads to only two possible options: either sur-
gical bypass revascularization or limb amputa-
tion. We investigated a novel method using
autologous transplantation of progenitor cells
derived from mobilized peripheral blood bone
marrow mononuclear cells to evaluate its long-
term effect as a cell therapy to induce neo-an-
giogenesis and restore blood flow in the af-
fected ischemic limbs. A total of 20 ischemic
limbs from critical limb ischemia diagnosed pa-
tients, non candidates to surgical revasculariza-
tion were transplanted with autologous proge-
nitor cells by either intramuscular combined
with intravenous (group A) or intramuscular
(group B) procedure. Patients were monitored
during 31 months. Treatment efficacy was eva-
luated according to the following parameters:
ankle brachial index which increased at a range
of 0.29 - 1 . 0 i n g r ou p A a nd 0. 4 0 - 0.90 in group B;
pain-free walking distance which increased at a
range of 50 - 600 m in group A and 50 - 300 m in
group B; and blood perfusion (measured by La-
ser Doppler) which increased at a range of 48 -
299 in group A and 135 - 225 in group B. We
achieved 90% treated ischemic limbs free of
amputation in both transplanted groups. Results
here described provide a safe, efficient and mi-
nimally invasive therapy with progenitor cells to
induce angiogenesis and preserve limbs from
amput ation in CLI diagnosed patients.
Keywords: Neo-Angiogenesis; Cell therapy; Critical
Limb Ischemia; Progenitor Cells; Blood Perfusi on
1. INTRODUCTION
Peripheral arterial disease (PAD) is a medical problem
that comprises the obstruction of blood flow in the arte-
ries causing inadequate oxygen supply to diverse tissues.
This disease currently affects approximately 12 million
people in the United States of America (USA) [1]. The
death risk for people with PAD is 10 to 15 times greater
than the death risk for subjects free from this disease as
shown in a 10 years follow up study [2]. PAD in lower
extremities is mainly expressed with intermittent claudi-
cation (IC) which is an early manifestation of pain during
ambulation; as disease progresses pain is presented even
at rest and patients usually develop a blood flow decrease
which leads to ischemic ulcerations and in late stages

L. Padilla et al. / Stem Cell Discovery 2 (2012) 155-162
156
gangrene requiring minor or major amputations, thus
completing the natural story for critical limb ischemia
(CLI) [3-5]. Diabetic patients are at a higher risk of de-
veloping such CLI condition, since 30% of them will
present it earlier in life, compared with non-diabetic
population [6,7]; besides, PAD in diabetes mellitus dis-
ease is more diffuse and severe, mainly affecting the
lower extremities vasculature, while the non-diabetic
patients with PAD have a higher aorto-iliac incidence
[8,9]. CLI is a growing medical problem and has an es-
timated incidence of 11% in the general population and
15% in adults over 55 years old; it also has been reported
with an incidence of 500 to 1000 individuals per million
each year [10]. The reported mortality rates due to CLI
has been 20%, 35% 70% and up to 100% for 1, 2, 5 and
10 years respectively and at least 50% of patients will
undergo major limb amputation within 6 to 12 months
[11-13]. Current treatments for CLI patients include se-
rum lipid levels reduction, antiplatelet (i.e. cilostasol)
and antihypertensive drugs showing limited efficacy in
CLI severe stages [14,15]. Although surgical revascu-
larization remains as the most appropriate CLI therapy
aimed to prevent limb loss, it is not suitable for at least
30% of patients because of extent of the disease and the
lack of proper non damaged autologous vasculature;
which leaves amputation as the only option if CLI has
progressed beyond the point of salvage, vascular surgery
is too risky or life expectancy is very low [16,17]. Inclu-
iding only USA 100,000 major limb amputations are
performed every year due to PAD with an annual cost of
more than 13 billion US dollars [18]. Patients with CLI
who are not eligible for revascularization have no effec-
tive treatment option. Despite there is no Federal Drug
Administration (FDA) approved therapy for these CLI
patients, there has been several research groups reporting
different strategies towards the establishment of pro-
genitor cell therapies to allow the formation of new
blood vessels (neo-angiogenesis) as a method to salvage
ischemic limbs through autologous transplantation of
progenitor cells derived from Bone Marrow-Mononu-
clear Cells (BM-MNC) [18-23]. At least 40 research
groups have obtained promising results with BM-MNC
transplantation as progenitor cell therapy treatment for
CLI patients, supporting evidence to establish this pro-
cedure as an alternative to improve blood perfusion
through neo-angiogenesis and to avoid amputation [3,
18]. To reach a maximum local cell concentration, Bartsch
et al. [21], performed 13 intramuscular and intra-arterial
transplantation of BM-MNC obtained from iliac crest
bone marrow aspiration and purified through ficoll gra-
dient technique, this procedure resulted in the improve-
ment of the ankle brachial index (ABI); in contrast, the
12 patients in the control group with no cell transplant
showed a statistically significant reduction of ABI and
venous occlusion plethysmography at rest. Using a rat
ischemic limb model we previously demonstrated the
induction of effective neo-vascularization after bone
marrow mononuclear cell (CD34+ and CD133+) trans-
plantation into surgically induced fibrocollagenous tun-
nels used as scaffolds to enhance cell survival and dif-
ferentiation [24]. In a second experimental study, our
group used dogs as an ischemic limb model confirming
that transplantation of mobilized BM-MNC to peripheral
blood through the use of Granulocyte Colony-Stimula-
ting Factor (G-CSF) statistically significant increased
angiogenesis as compared with cell transplant without
G-CSF treatment [25]. Based on the evidence of neo-an-
giogenesis as a result of mobilized BM-MNC autologous
transplantation on these experimental models, we ob-
tained the ethical and research institution committee ap-
proval to start a clinical trial in humans for the progenitor
cell therapy of CLI patients. In this report we analyze the
efficacy of Mobilized BM-MNC transplantation com-
paring combined procedure intramuscular and distal ret-
rograde-intravenous (saphenous vein) transplantation
(IM + IV) versus intramuscular (IM) standard transplan-
tation procedure.
2. MATERIALS AND METHODS
2.1. Trial Profile
The institutional board approved this prospective, con-
trolled, randomized study; all enrolled patients provided
written informed consent; all surgical procedures were
conducted by senior expert surgeon (PL and RTJ).
2.2. Patient’s Inclusion Criteria
Patients older than 18 years old presenting severe
lower limb obstructive arterial disease classified as
Fontaine stage IIb (Incapacitant intermittent claudica-
tion), Fontaine stage III (pain at rest), Rutherford 4, with
an ankle-brachial index (ABI) lower than 0.6, and who
were non candidates to surgical bypass revascularization
nor to endovascular procedure.
2.3. Patient’s Exclusion Criteria
Patients with any neoplasia history in the last 10 years,
patients with 1 year of life expectancy, patients with se-
vere renal failure, severe malnutrition, or systemic chronic
infectious disease at any clinical stage (Hepatitis B, C or
HIV infection).
The parameters to evaluate were procedure’s safety,
treatment efficacy, ABI, blood perfusion and pain-free
walking distance evolution. Patients of both experimental
and control groups were monitored by measuring those

L. Padilla et al. / Stem Cell Discovery 2 (2012) 155-162 157
parameters at 0, 3, 12 months after progenitor cell trans-
plant and at 18, 24 and 31 months after cell transplant
therapy to evaluate the ratio of patients free of limb am-
putation.
2.4. Study Design
The study was designed as a randomized prospective
controlled clinical trial and its main goal was to improve
blood perfusion through neo-angiogenesis and to salvage
patient’s ischemic limbs. 20 critical ischemic affected
limbs (Fontaine II-b, Fontaine III and Rutherford 4) were
evaluated in 14 patients non candidates to any form of
revascularization who accepted to participate in the study
and were included in the trial according to the estab-
lished inclusion criteria. Limb ischemia diagnosis was
based on Laser Doppler blood flow measurements (≤31
perfusion units), ankle brachial index (≤0.6) and pain-
free walking distance (≤200 m). Patients were randomly
divided into two main groups according to the following
transplant strategy (as shown in Figure 1):
Group A: 10 ischemic limbs from patients transplanted
with progenitor cells using a combination of intramus-
cular and distal retrograde-intravenous (saphenous vein)
transplant. Group A is referred in this report as IM + IV
(Intramuscular + Intravenous).
Group B (control group): 10 ischemic limbs from pa-
tients with progenitor cell intramuscular transplant. Group
B is referred in this report as IM (intramuscular).
2.5. Cell Harvest
Mobilization of Bone Marrow-Mononuclear Cells (B-
M-MNC) to peripheral blood was achieved by a daily
subcutaneous administration of 5 μg of Granulocyte Co-
lony-Stimulating Factor (G-CSF) (Roche) during 5 con-
secutive days at early morning. Mobilized Peripheral
Blood Mononuclear Cells (M-PBMNC) were harvested
on the 5th day of treatment by apheresis procedure using
a continuous flow COBE-Spectra blood cell automated
separator (COBE BCT, Lakewood CO., USA) and a dou-
ble puncture technique using citrate plus glucose as an-
ticoagulant and physiologic solution as primer. Aphe-
resis was performed as a two-step procedure involving
extraction and re-infusion using a Mahurkar or Niagara
catheter and extraction/infusion flow speeds between 50 -
55 mL·min−1. For all patients a 60 mL standard amount
of progenitor cell suspension per ischemic limb was ob-
tained at the end of the apheresis procedure.
2.6. Cell Analysis
CD34+ (hematopoietic) and CD133+ (angioblast) mouse
anti human antibody (Beckton Dickinson, Ca, USA) cell
marker were used to confirm progenitor cell phenotype-
respectively by flowcytometry analysis using a FAC
Figure 1. Study design. A total of 14 patients were re-
cruited for this study achieving 10 ischemic limbs per
group. Some of the patients presenting both ischemic limbs
were included as one of the ischemic limbs in group A
and the second ischemic limb in group B.
SCalibur and Cell Quest Pro Software (Becton Dickinson,
Ca, USA). Cell count was manually obtained using a
haemocytometer.
2.7. Surgical Technique
Ultrasound guided punctures on the affected limb
muscle compartments were performed under epidural
blockade and light sedation using a catheter needle BD
Insyte (16GA-1.77IN) (17 × 45 mm). The 60 mL
M-PBMN cell suspension per ischemic limb were dis-
tributed as follows: 10 mL in the internal vastus com-
partment, 10 mL in the external vastus compartment, 10
mL in the anterior tibial muscle compartment, 10 mL in
the deep posterior muscle compartment and 10 mL in the
superficial posterior muscle compartment. For group A,
as part of the surgical procedure, the major saphenous
vein was localized at the internal malleolus level, at this
site a small incision was performed in order to dissect the
vein and isolate it using chromic catgut 3 - 0 sutures. Af-
terwards the vein was temporarily ocluded in a proximal
direction and a catheter BD Insyte (16GA-1.77IN) (17 ×
45 mm) was placed by direct puncture in order to per-
form a regional distal retrograde heparinization (1 mL of
heparine diluted 1:1000 + 9 mL saline solution). A ma-
nual tourniquet was applied to avoid the venous flow to
return for 10 minutes followed by the distal retrograde
intravenous application of the last 10 mL cell suspension
(Figure 2). For group B, 12 mL instead of 10 mL of M-
PBMN cell suspension were distributed between the 5
muscles compartments as previously mentioned.

L. Padilla et al. / Stem Cell Discovery 2 (2012) 155-162
158
Figure 2. Surgical technique used to induce neo-angioge-
nesis. Cell transplant was performed ultrasound guided
(1), cells were transplanted at different muscle compart-
ments such as: internal and external vastus compartments,
anterior tibial muscle compartment (2A), deep posterior
muscle compartment (2B), superficial posterior muscle
compartment (2C) and at internal malleolus level (3 and 4).
2.8. Lasser Doppler Technique
Blood flow by Laser Doppler measurement is a stan-
dard technique for real time measurement of tissue per-
fusion at micro-vascular level. This technique was first
reported in 1964 by Yeh and Cummins [26], and the units
of Laser Doppler are given in tissue-Perfusion Units
(PU); such PU was standardized through the use of a
suspension of latex spheres in movement. This method is
non invasive, highly sensitive to local perfusion and
useful for continuous monitoring if necessary. A signal
proportional to red blood cells (RBC) perfusion is dis-
played in the screen of the Laser Doppler equipment; this
signal represents the transport of RBC through mi-
crovasculature [27]. Patient’s blood flow measurements
were performed by Lasser Doppler technique using a
Periflux System 5000 (Perimed Instruments, Stockholm
Sweden) prior to the transplant procedure and at 3 and 12
months post transplant in both groups. Lasser Doppler
measurements were performed for at least 2 minutes for
baseline values prior to temperature increase as stimulus
to record RBC perfusion units (See Figure 3).
2.9. Statistical Analysis
A database corresponding to quantitative baseline and
post treatment values for blood perfusion and ABI per
patient was used for statistical analysis. Changes from
baseline to 3 and 12 months variables were analyzed by
paired t-test according to normal distribution. Statistical
significance was assumed for p < 0.05 value. All statis-
ticcal analysis was performed with SPSS 16.0 statistical
software.
3. RESULTS
3.1. Clinical Characteristics
Fourteen patients with severe CLI (Fontaine IIb, Fon-
taine III, Rutherford 4) were recruited, transplanted,
evaluated and monitored during 31 months. Clinical cha-
racteristics from recruited patients are shown in Table
1(a) and Table 1(b) shows ABI and pain free-walking dis-
tance values prior to treatment (defined as time zero—t0)
and post treatment at 12 months (t12).
(a)
(b)
Figure 3. Representative blood flow measured by Laser
Doppler prior to transplant (a) and 12 months post transplant
(b).
Table 1. (a) Patient clinical characteristics; (b) Clinical follow
up after 12 months post transplantation.
(a)
Clinical
Characteristic Group A (IM + IV) N = 10 Group B (IM) N = 10
Smoking 8 5
Hypercholesterolemia5 6
Diabetes Mellitus 6 2
Arterial Hypertension 8 6
Myocardial Infarct 2 2
(b)
Parameter Group A (IM + IV) Group B (IM)
Limb Amputation 1* 1
**
Pain-Free Walking
Distance (m) Range
(min - max)
50 - 600 50 - 300
ABI Range
(min - max) 0.29 - 1.0 0.40 - 0.90
Blood Flow (PU)
Range (min - max) 48.82 - 299.02 135.13 - 225.31
*Limb amputation on the 8th month post transplant; **Limb amputation
on the 6th month post transplant.

L. Padilla et al. / Stem Cell Discovery 2 (2012) 155-162
159
3.2. Transplantated Cell Number
Progenitor cell phenotype was identified by flowcytome-
try with CD34+ and CD133+ cell markers (as previously
described). The total transplanted cell number per is-
chemic limb were 17.4 ± 8 × 106 CD34+ cells for group
A, and 15 ± 5 × 106 CD34+ cells for group B; meanwhile
the total transplanted CD133+ cells for group A were 2.86
± 5.96 × 107 and 3.01 ± 5.91 × 107 for group B.
3.3. Effect of Transplantated Progenitor
Cells
Patients were monitored at 0, 3, and 12 months post
progenitor cell transplant; parameters evaluated included:
Pain free walking distance (reported in meters), ABI,
blood flow reported as perfusion units, angiography and
ischemic limbs free of amputation (monitored during
31 months post transplant). At 31 months post treatment
period it was reported 1 limb amputation in each group,
A and B (Tables 1(b) and 2), interestingly both patients
were diabetics. Average values for pain-free walking dis-
tance parameter increased from 60 ± 39 m pre-transplant
to 207 ± 198 m at 12 months post-transplant for group A,
and from 87 ± 69 m pre-transplant to 172 ± 127 m at 12
months post-transplant for group B. ABI parameter
showed an increase from 0.42 ± 0.14 pre-transplant to
0.70 ± 0.21 at 12 months post-transplant for group A, and
from 0.44 ± 0.09 pre-transplant to 0.62 ± 0.15 at 12
months post-transplant for group B. Blood perfusion
(measured by Laser Doppler) increased from15.8 ± 9.3
pre-transplant to 205.8 ± 116.7 at 12 months post-trans-
plant for group A, and from 16.6 ± 7 acute myocardial
infarction. There were no reported adverse effects due to
cell transplant procedure. Blood flow perfusion units
Table 2. Baseline and monitored clinical parameters.
Extremity Limb Age
(years) Gender ABI t0 ABI t12
Pain free
walking
distance
t0
(Meters)
Pain free
walking
distancet12
(Meters)
Blood
perfusion
(t0)
Blood
perfusion
(t3) Observation
Group A (IM + IV)
1 L 77 F 0.70 1 20 100 Salvage
2 R 72 M 0.40 0.50 100 150 6.46 13.53 Salvage
3 U 79 F 0.20 0.80 No dataNo data 8.99 30.53 Salvage
4 U 59 M 0.50 0.70 No dataNo data 8.23 6.10 Amputate
5 R 67 F 0.86 0.93 100 350 14.07 17.23 Salvage
6 R 54 F 0.50 0.50 50 50 30.60 38.27 Salvage
7 R 63 M 0.26 0.55 10 100 30.56 42.10 Salvage
8 U 70 F 0.40 0.80 No dataNo data 10.56 39.20 Salvage
9 R 68 M 0.36 0.70 40 600 20.20 No data Salvage
10 L 64 F 0.50 0.29 100 100 12.70 31.00 Salvage
Group B (IM)
1 R 67 M 0.50 0.50 150 1200 7.4 17.26 Salvage
2 L 67 M 0.50 0.60 150 1200 14.1 31.20 Salvage
3 L 72 M 0.40 0.60 100 150 14.49 16.09 Salvage
4 L 54 F 0.60 0.50 50 50 8.97 52.38 Salvage
5 R 46 F 0.50 0.70 200 400 23.00 61.84 Salvage
6 R 70 F 0.40 0.80 10 50 18.70 37.40 Amputate
7 R 64 F 0.50 0.40 100 100 15.61 24.15 Salvage
8 R 63 M 0.26 0.50 10 100 30.56 42.10 Salvage
9 R 77 F 0.40 0.90 20 100 No data No data Salvage
10 R 78 M 0.40 0.70 No dataNo data No data No data Salvage

L. Padilla et al. / Stem Cell Discovery 2 (2012) 155-162
160
measured by Laser Doppler (as described in Materials
and Methods), showed a significant improvement by an
average of 14 fold increase (p < 0.05) in the case of group
A and a 10 fold increase (p < 0.05) for group B as com-
pared with the corresponding baseline blood flow meas-
urements for each group. These results suggest that de-
spite both transplant procedures resulted in an important
blood flow increase, the effect was higher when pro-
genitor cells were transplanted using a combined intra-
muscular-intravenous method. Figure 4 shows blood
perfusion from 8 critical ischemic limbs at pre transplant,
at 3 and 12 months post transplant as evidence on the
increase of blood perfusion after treatment for both
groups Figure 5 shows average perfusion unit values for
group A and group B pre and post transplant. An-
giographic base line (time zero) index and blood per-
fusion at 12 months time point, were not performed in all
patients due to logistic follow up limitations, however at
least 6 patients were subjected to angiography.
Figure 6 shows angiography pre and post transplant
for one of the patients included in group A and one in the
group B as an example of neo-angiogenesis due to pro-
genitor cell transplant. The neo-angiogenesis process was
confirmed with the increase in the blood flow perfusion
as shown in Figures 4 and 5. Despite ABI, pain free walk-
ing distance and blood perfusion parameters were not
measured in most of patients at 24 and 31 months, the
beneficial effects of treatments were maintained as no
limb amputations were performed.
4. DISCUSSION
In this study, we found that CLI diagnosed patients
treated by any of both procedures, either A or B pro-
genytor cell transplanted groups, induced neo-angio-
genesis improving all clinical parameters and avoiding
limb amputation as 90% of limbs in each group were
Figure 4. Blood flow given as perfusion units at pre transplant,
3 and 12 months post transplant.
Figure 5. Average PU values for group A and group B.
Comparison of blood perfusion between baseline values (pre
transplant) and 12 months post transplant resulted in statis-
tically significant differences for (dashed line) group A (p <
0.05) and (dotted line) group B (p < 0.05).
Figure 6. Angiography performed at
pre-transplant and 12 months post trans-
plant. Angiography for: (a) one patient
from group A and (b) one patient from
group B.
saved. It was found a higher efficiency in terms of blood
perfusion, ABI and pain-free walking distance when cell
transplant was performed with a combined intra mus-
cular and intra venous procedure; however there was no
statistical significant difference between groups. We pre-
viously performed the transplant of progenitor cells into
surgically induced fibrocollagenous tunnels to a total of
8 ischemic limbs; we initially observed a significant cli-
nical improvement in 5 of the ischemic limbs while the
other 3 showed non favorable clinical outcome with im-
provement in vascular proximal blood perfusion but de-
veloped necrosis in toes (non published data). We con-
cluded that for those 3 patients the transplant technique
did not allow transplanted cells to reach toe’s capillaries.
Based on these results, we developed a new protocol in
CLI induced rats (Sprague Dawley) as a model in order
to allow transplanted cells to reach toe’s capillaries; we
demonstrated that by adding a distal retrograde-intrave-
nous injection of cells, higher levels of angiogenesis

L. Padilla et al. / Stem Cell Discovery 2 (2012) 155-162 161
were produced as compared with intramuscular cell
transplant. We also observed that mononuclear cells attach-
ed to the smallest capillary segment used as a scaffold
increased survival, proliferation, and cell differentiation
towards endothelial cell type [28]. Tepper [29] and Law
all [30] demonstrated that angiogenesis or vasculogene-
sis induction requires the ischemic initial stimulus and
the muscle inflammatory response that releases cytokines
and growth factors (such as VEGF, bFGF, SDF-1, PDG-
BB, IGF-1, TGF-B) that activate proliferation, migration
and tubulization of endothelial cells. These findings
support the use of muscle as the main target for progeny-
tor cell transplant, however we showed that combined
intramuscular and intravenous transplant technique pro-
vides a higher transplant efficiency as demonstrated in
our previous animal model report [26]. A similar 12
months follow up study reported by Van Tongeren et al.
[31] demonstrated a higher efficiency when combined
intramuscular and intra-arterial transplant strategy was
used in comparison with only intramuscular transplant.
The main results of our study is the evidence that both
transplant procedures increases blood perfusion, ABI and
pain free walking distance; showing slightly higher re-
sults for combined intramuscular-intravenous group. More
importantly, improvements achieved by progenitor cell
transplant in this study, were maintained for up to 31
months achieving 90% of the limbs preserved from am-
putationn.
5. CONCLUSION
Progenitor cell therapy described in this study is a
minimally invasive therapeutic option for patients with
critical limb ischemia. Both, intramuscular and com-
bined intramuscular-intravenous autologous progenitor
cell transplant led to a substantial increase in blood flow
perfusion, pain-free walking distance and ankle brachial
index. Despite there was no statistical significance be-
tween groups, a higher blood perfusion (measured by
laser Doppler) was observed when progenitor cells were
transplanted combining intramuscular-intravenous as com-
pared with intra muscular procedure. Transplant of pro-
genitor cells described here provides a safe and reliable
cell therapy for CLI diagnosed patients who are not can-
didates for invasive surgical revascularization.
6. ACKNOWLEDGEMENTS
We would like to acknowledge to Reneé Acosta and Paola Lares
Juárez for their help on the revision of the manuscript.
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