Allogeneic transplantation of programmable cells of monocytic origin (PCMO) improves angiogenesis and tissue recovery in critical limb ischemia (CLI): a translational approach
Berndt et al. Stem Cell Research & Therapy
Allogeneic transplantation of programmable cells of monocytic origin (PCMO) improves angiogenesis and tissue recovery in critical limb ischemia (CLI): a translational approach
Rouven Berndt 0 1
Lars Hummitzsch 0 3
Katharina Heß 0 2
Martin Albrecht 3
Karina Zitta 3
Rene Rusch 1
Beke Sarras 1
Andreas Bayer 1
Jochen Cremer 1
Fred Faendrich 4
Justus Groß 1
0 Equal contributors
1 Department of Cardiaovascular Surgery, University Hospital of Schleswig-Holstein , Campus Kiel, Arnold-Heller-Str. 3, Hs 18, D-24105 Kiel , Germany
2 Institute of Neuropathology, University Hospital Münster , Münster , Germany
3 Department of Anesthesiology and Intensive Care Medicine, University Hospital of Schleswig-Holstein , Kiel , Germany
4 Department of Applied Cell Therapy, University Hospital of Schleswig-Holstein , Kiel , Germany
Backround: Employing growth factor-induced partial reprogramming in vitro, peripheral human blood monocytes can acquire a state of plasticity along with expression of various markers of pluripotency. These so-called programmable cells of monocytic origin (PCMO) hold great promise in regenerative therapies. The aim of this translational study was to explore and exploit the functional properties of PCMO for allogeneic cell transplantation therapy in critical limb ischemia (CLI). Methods: Using our previously described differentiation protocol, murine and human monocytes were differentiated into PCMO. We examined paracrine secretion of pro-angiogenic and tissue recovery-associated proteins under hypoxia and induction of angiogenesis by PCMO in vitro. Allogeneic cell transplantation of PCMO was performed in a hind limb ischemia mouse model in comparison to cell transplantation of native monocytes and a placebo group. Moreover, we analyzed retrospectively four healing attempts with PCMO in patients with peripheral artery disease (PAD; Rutherford classification, stage 5 and 6). Statistical analysis was performed by using one-way ANOVA, Tukey's test or the Student's t test, p < 0.05. Results: Cell culture experiments revealed good resilience of PCMO under hypoxia, enhanced paracrine release of pro-angiogenic and tissue recovery-associated proteins and induction of angiogenesis in vitro by PCMO. Animal experiments demonstrated significantly enhanced SO2 saturation, blood flow, neoangiogenesis and tissue recovery after treatment with PCMO compared to treatment with native monocytes and placebo. Finally, first therapeutic application of PCMO in humans demonstrated increased vascular collaterals and improved wound healing in patients with chronic CLI without exaggerated immune response, malignant processes or extended infection after 12 months. In all patients minor and/or major amputations of the lower extremity could be avoided. Conclusions: In summary, PCMO improve angiogenesis and tissue recovery in chronic ischemic muscle and first clinical results promise to provide an effective and safe treatment of CLI.
Programmable cells of monocytic origin; Monocytes; Stem cells; Peripheral arterial disease; Critical limb ischemia; Cell therapy; Regenerative medicine
Critical limb ischemia (CLI) results from insufficient
supply of blood due to arterial stenosis/ occlusion, vessel
trauma or vasoconstriction because of catecholamine
therapy and shock. Incidence of CLI is ~ 220 new cases
per 1 million per year and 30% of these patients will not
have options in open or endovascular revascularization
]. For these patients, major limb amputation
remains as the only lifesaving treatment option. In
response to the significant need for new strategies to
prevent tissue damage after CLI and consecutive major
limb amputation, there have been numerous studies
investigating different strategies in cell-based therapy for
inducing neoangiogenesis in ischemic tissues. These
previous studies mostly investigate the application of bone
marrow-derived hematopoietic stem cells in ischemic
tissue but also include a wide range of embryonic stem
cells, mesenchymal cells, skeletal myoblasts, and
endothelial progenitor cells [
Among them, dedifferentiated progenitor cells of
myeloid precursors have been described as a promising
strategy for cell transplantation, inducing regeneration of
chronic ischemic tissue [
]. However, most
approaches in cell therapy have yet failed to generate a
significant impact on clinical practice because of several
unsolved issues [
]. First, transplanted cells are
generally insufficiently assumed in ischemic tissue and
longtime resilience is usually not featured and
investigated in angiogenic therapy. Second, usually a high
number of cells for cell transplantation and thus a
feasible source in clinical practice is necessary. Third, in
most cases transplanted cells may not have been directly
involved in forming vascular structures but possibly
contribute to neoangiogenesis and vascular remodeling by
paracrine mechanisms, which have not been investigated
in detail so far [
Considering these aspects, programmable cells of
monocytic origin (PCMO) provide several characteristics
rendering them potentially valuable for solving the major
limitations of cell transplantation therapy in CLI. As
recently reported by our group, peripheral blood
monocytes can be partially dedifferentiated into CD14+
progenitor cells with multipotent properties resuming
cell division and downregulated factors known to
promote their terminal differentiation while retaining other
characteristics of the myelomonocytic lineage [
PCMO have been reported to provide strong expression
of CD14, CD90 and CD115 and weak expression of
CD123 whereas other monocyte and stem cell markers
such as CD16, CD34, CD117 and CD135 were low or
]. Dedifferentiated monocytes have
been shown to be responsive to inductive stimuli that
directed differentiation into somatic cells of all three germ
]. Successful tissue engineering from PCMO as
well as significant improvement of ischemic heart
muscle in animal studies after treatment with PCMO
has supported cautious optimism that multipotent
dedifferentiated monocytes could also be used for angiogenic
Based on our previous research [
11, 15, 16
hypothesized that PCMO can contribute to
neoangiogenesis and recovery of ischemic muscle in a paracrine
manner. Here, we describe a translational approach and
provide evidence from (1) in vitro experiments, (2)
animal studies and (3) first healing attempts in men, that
PCMO exert therapeutic pro-angiogenic effects and
might be suitable for the treatment of CLI.
Isolation and generation of PCMO from mouse and human origin
Mononuclear cells were isolated from peripheral blood
of male C57BL/6 donor mice by (Charles Rivers
Laboratories, Wilmington MA, USA) density gradient
centrifugation. Cells (1.3 × 107/cm2) could adhere to the bottom
of tissue culture flasks for 1 to 2 h in RPMI medium
containing 10% fetal calf serum (FCS), 2 mM glutamine,
100 U/mL penicillin and 100 mg/mL streptomycin (all
from Invitrogen, Karlsruhe, Germany). Nonadherent
cells were removed by gentle washing with
phosphatebuffered saline (PBS) and were cultured for 4 days in
‘dedifferentiation medium’ consisting of RPMI 1640-based
medium with 140 μM ß-mercaptoethanol, 20 ng/ml
murine monocyte/macrophage colony-stimulating
factor (M-CSF) (R&D Systems, Wiesbaden, Germany)
and 0.4 ng/ml murine interleukin (IL)-3 (R&D
Systems). On day 4, the cells (from now termed PCMO)
were washed with PBS and harvested mechanically.
Characterization of cell lineage was performed by flow
cytometry. Antibodies were directly conjugated with
either phycoerythrin (CD3, CD34, CD45, CD80, CD86,
CD90, and CD117, all from Beckmann Coulter, Krefeld,
Germany; CD13, CD123, CD135 from BD, Heidelberg,
Germany), FITC (CD19, Beckmann Coulter), or
PC5/PECyTM5 (CD14, Beckmann Coulter).
Human PCMO were generated by leukapheresis
products from five healthy donors (D1–5) and provided by
the Department of Transfusion Medicine (University
Hospital Schleswig Holstein, Kiel). Mononuclear cells
were isolated by density gradient centrifugation (1.3 ×
107/cm2) and allowed to adhere to the bottom of tissue
culture flasks for 1 to 2 h in RPMI medium 1640
containing 10% human AB-Serum (Lonza, Verviers,
Belgium), 2 mM glutamine, 100 U/ml penicillin and
100 mg/ml streptomycin (all from Invitrogen, Karlsruhe,
Germany). Nonadherent cells were removed by gentle
washing with PBS and cultured for 4 days in
‘dedifferentiation medium’ consisting of RPMI-based medium with
140 μM ß-mercaptoethanol, 5 ng/ml human M-CSF
(R&D Systems, Wiesbaden, Germany) and 0.4 ng/ml
human IL-3 (R&D Systems). On day 4, the cells were
washed with PBS and harvested mechanically.
Characterization of cell lineage was also performed by
flow cytometry as previously reported [
]. PCMO for
clinical application were generated in accordance with
the European Union, Good Manufacturing Practice (EU
Induction of hypoxia in PCMO cell cultures
Simulation of ischemic conditions in vitro was
performed by using our recently described enzymatic
hypoxia model [
] (Fig. 1a). A series of pretests prior to
the major experiments evaluated the optimized duration
of transient hypoxia (3 h) and the observation interval
(24 h) considering the increase of ischemia-inducible
factors in PCMO (Additional file 1: Figure S1) and
progression of cell damage during CLI [
Employing glucose oxidase (Sigma-Aldrich,
Schnelldorf, Germany; final concentration 4 U/ml) and catalase
(Sigma-Aldrich, Schnelldorf, Germany; final
concentration 240 U/ml) in DMEM high-glucose medium with 1%
FCS (PAA, Coelbe, Germany) in combination with a
standard six-well system (NUNC, Roskilde, Denmark),
partial pressure of oxygen (pO2) in the culture medium
and its temporal decline after the addition of glucose
oxidase and catalase was measured by using a flexible
probe (LICOX® CMP Oxygen Catheter, Integra,
Plainsboro, NJ, USA). Concentrations of glucose within the
culture media were determined using the Fehling’s
method. Fehling’s reagents I and II (Sigma-Aldrich,
Schnelldorf, Germany) were mixed with the samples and
boiled in a water bath for 15 min. Absorbance was
determined at 495 nm using an enzyme-linked
immunosorbent assay (ELISA) reader (Tecan, Crailsheim, Germany)
with Magellan software v1.1. Standard curves were
created from known concentrations of glucose.
Isolation of RNA and polymerase chain reaction
Cells were washed twice with phosphate-buffered saline
(Sigma-Aldrich, Schnelldorf, Germany) and suspended
in RLT buffer. Isolation of RNA was done with the
Qiagen RNeasy minikit according to the manufacturer’s
protocol (Qiagen, Hilden, Germany). RNA
concentrations in the samples were quantified with a
spectrophotometer at 260 nm. Purity of RNA was assessed by the
260/280 nm ratio. A total of 200 ng total RNA was used
to produce cDNA by a reverse transcription kit (Applied
Biosystems, Carlsbad, CA, USA) using random hexamer
primers. A 2 μl sample was used as a template for PCR
experiments in a final volume of 20 μl. All PCR
experiments were performed with DNA Taq Polymerase from
Solis BioDyne (Tartu, Estonia). Primers were chosen
based on the available literature about ischemia-induced
gene expression in monocytes/macrophages [
(Additional file 1: Figure S1). The primer sequences are given
in an additional Table (Additional file 2: Table S1).
Negative controls were performed by omitting the respective
input cDNA. PCR products were separated on 2.5%
agarose gels followed by ethidium bromide staining and
were visualized by ultraviolet transillumination. For
evaluation of gene expression levels, gels were scanned
and the respective bands were densitometrically analyzed
with the software ImageJ (v1.41o; National Institutes of
Health, Bethesda, MD, USA). Values are depicted as
relative densitometric units.
LDH cytotoxicity assay
The colorimetric Cytotoxicity Detection KitPLUS (Roche,
Mannheim, Germany) was used for the quantification of
cell damage by measuring lactate dehydrogenase (LDH)
activity released from cultured cells (Additional file 3:
Figure S2). Preparation of samples and measurements
were performed according to the specifications of the
manufacturer. Briefly, cell culture supernatants were
collected 24 h after hypoxia. For the evaluation of
total LDH activity, cell lysis was performed with 2%
Triton X-100 (Roth, Karlsruhe, Germany). The 100-μL
samples were measured per well of a 96-well plate at
492 nm using an ELISA reader (Tecan, Crailsheim,
Austria) with Magellan software v1.1 (Tecan, Crailsheim,
Austria), and values of absorbance were depicted as
arbitrary units (a.u.).
Proteome profiling arrays
Proteome profiling arrays (R&D Systems, Minneapolis,
MN, USA) were performed according to the protocol of
the manufacturer. After culturing and treating PCMO as
described above (2.1), equal amounts (40 μg) of cell
lysates (for intracellular proteins) and 150 μl of cell
culture medium (for secreted proteins) from each donor
(n = 5) were pooled and applied to the respective array
membrane (Fig. 1a). Expression levels of 55 angiogenesis
and tissue recovery associated proteins were evaluated
by densitometric analyses of the arrays using the ImageJ
1.41 software (ImageJ, NIH, Bethesda, MD, USA).
Isolation of human umbilical vein endothelial cells (HUVEC)
Human umbilical vein endothelial cells (HUVEC) were
freshly isolated from umbilical cords as described
] and maintained in a humidified atmosphere
(5% carbon dioxide/95% air) at 37 °C in endothelial cell
growth medium (ECGM) (Promocell, Heidelberg,
Germany) supplemented with 4 μl/ml of endothelial cell
growth supplement, 0.1 ng/ml epidermal growth factor,
1 ng/ml basic fibroblast growth factor, 90 μg/ml heparin,
Berndt et al. Stem Cell Research & Therapy (2018) 9:117
Fig. 1 (See legend on next page.)
(See figure on previous page.)
Fig. 1 Experimental protocol includes (a) cell culture experiments of PCMO evaluating the potential pro-angiogenic effect in vitro, (b) animal
studies examining the potential effect of treatment with PCMO in contrast to application of native monocytes/placebo group and (c) a series of
individual healing attemps in patients with peripheral arterial disease (Rutherford, stage 5 and 6) without further curative treatment options.
HUVEC human umbilical vein endothelial cells, PCMO programmable cells of monocytic origin
1 μg/ml hydrocortisone (all from Promocell) and 10%
heat-inactivated fetal bovine serum (Thermo Fisher,
Schwerte, Germany), and maintained as primary cell
culture. For angiogenesis, cells were detached with a mild
cell detachment solution (Accutase, San Diego, CA,
USA) and seeded as indicated for the experiments.
Endothelial tube formation with PCMO-conditioned cell culture supernatants
HUVEC were resuspended in the respective
PCMOconditioned cultured media (2 × 105cells/ml), and 50 μl
were seeded into angiogenesis imaging chambers (Ibidi
GmbH, Muenchen, Germany) containing Matrigel (Fig.
1a). After 2 h, photomicrographs of the cells were taken
every hour until 7 h. The analyses compared PCMO
supernatants hypoxia-conditioned vs. PCMO
supernatants non-hypoxic-conditioned from five donors (D1–5)
and pooled samples from all donors. Angiogenesis
parameters (number of segments, nodes and meshes) from
each picture were evaluated with the angiogenesis
analyzer of the ImageJ software (v1.410) at six different
time points (2 h–7 h), as previously described [
To evaluate whether PCMO induce neoangiogenesis in
vivo, a hind limb ischemia mouse model was introduced
for the evaluation of putative paracrine effects of the
PCMO prior to the first clinical application (Fig. 1b).
Seven-week-old male C57BL/6 mice (Charles Rivers
Laboratories, Wilmington MA, USA) weighing 26 ± 4 g
were housed individually with fresh food and water ad
libitum during the holding time. To avoid transport
stress, the animals had been observed for 10 days before
the series of experiments began. All mice underwent
surgery and consecutive hind limb ligature and were
distributed among three experimental groups: the first
group (n = 24) was treated with PCMO (1 × 106 cells),
the second group (n = 24) was treated with native
monocytes (1 × 106 cells) and the third group (n = 24)
was the placebo group without treatment.
Hind limb ischemia was induced in mice using a slight
modification of a previously described method [
simulate long-term ischemia and to avoid complete
necrosis of the hind limb. All animal experiments were
performed under ketamine/xylasine anesthesia (100/
16 mg/kg body weight). Microsurgical-trained physicians
performed all surgical procedures. After the hind limbs
were shaved and cleaned (with 70% ethanol and betadine),
a longitudinal skin incision of ~ 1 cm was made in the
groin region, the connective tissue was removed and the
femoral artery, vein and nerve were exposed. Two 7/0
polypropylene ligatures were applied on the femoral artery
between the distal branch of the artery epigastric
superficialis and the trifurcation of the femoral artery into the
descending genicular, popliteal, and saphenous branches.
Mice were then allowed to recover under a heating lamp
to prevent hypothermia. For analgesia, all animals received
subcutaneous injections of buprenorphine hydrochloride
(Temgesic®, 0.1 mg/kg body weight) prior to surgery and
on the first postoperative day. One day after surgery,
allogeneic cell transplantation (1 × 106 cells/ 2 ml aqua ad)
was performed by intramuscular (i.m.) injection at the
anteromedial and posterior muscle compartment of the
left thigh. Animals were visually examined for toe necrosis
and/or toenail loss by a blinded observer at intervals of 7
days and for a total of 91 days.
Microlight guide spectrophotometry (O2C)
In vivo characterization of microcirculation in mice was
performed by micro-lightguide spectrophotometry
(O2C; LEA-Medizintechnik GMBH, Gießen, Germany),
as previously described [
]. Oxygen saturation (SO2)
and capillary blood flow was noninvasively assessed by
O2C at intervals of 7 days and for a total of 91 days.
Animals were narcotized with ketamine/xylasine as
described in 2.1 and fixed on the back with outstretched
legs. Measuring points were marked to ensure
reproducibility of the measurements. Probes were positioned
between the thighs extensor, adductors and lower leg
extensor (Fig. 1b). Pretests (not shown) were performed to
evaluate and avoid inter-observer variability. The
Doppler data were normalized as the mean ratio of ischemic
to contralateral limb measurements.
Histological and immunofluorescence analysis
Paraffin-embedded tissue slides with thickness of 5 μm
from the hind limbs of the mice were stained with
hematoxylin-eosin (HE) and myoglobin (1:1000,
DakoCytomation, Glostrup, Denmark) to evaluate the
morphometrics of the ischemic muscle (Fig. 1b).
Immunohistochemistry was carried out using the
avidin-biotin-peroxidase technique. Antibodies against
CD34 (1:200, Abcam, Cambridge, United Kingdom) and
CD105 (1:4000, Abcam, Cambridge, United Kingdom)
were applied to identify emerging endothelial vascular
tubes, capillaries and arterioles, as previously described
]. The secondary antibody was a mixture of
antirabbit and anti-goat mouse sera. Diaminobenzidine
(Leica Biosystems, Nussloch, Germany) served as
chromogen. Vascular density (VD) as well as tissue degeneration
(decrease of muscle fibers and increase of steatosis) were
determined by scanning ten randomly selected high power
fields (HPF) at ×400 magnification and in a blinded
manner. Positive capillaries (CD105 staining) and arterioles
(CD34 staining) were quantified separately and multiplied
to the number of positive cells in the same HPF. This
resulted in two distinct scores, presented as mean ± SD.
Steatosis was expressed as percentage of adipocytes versus
the total number of cells per HPF. Muscle degeneration
was expressed as number of muscle fibers per mm .
Retrospective analysis of four clinical individual treatments with PCMO in men
The Institutional Ethics Committee approved the first
therapy with PCMO in humans pursuant to clinical
individualized treatments in patients with chronic peripheral
artery disease (PAD; Rutherford classification, stage 5
and 6), ankle brachial index < 0.6, no option of surgically
revascularization and no further treatment option, except
major amputation (independently determined by two
senior surgeons). All clinical individualized treatments
were in accordance with the 1964 Helsinki declaration
and its later amendments or comparable ethical standards.
Individual patient consent for the individualized treatment
was obtained prior to therapy. PCMO for clinical
applications were generated in the Clinic of Applied Cell Therapy
(University Hospital Schleswig Holstein, Kiel, Germany)
in accordance with the EU GMP guidelines. PCMO (8 ×
108 cells/ 10 ml aqua ad) were injected 3.5 cm deep at 30
sites into the anteromedial and posterior muscle
compartment of the lower leg (Fig. 1c). For detailed
clinical presentation and demographic data, please refer to
additional Table S2 (Additional file 4: Table S2). All
patients were supervised in our outpatient unit. Aftercare
check-up followed at 4-week intervals and control
angiography was performed after 6 months (Fig. 1c). To evaluate
the effectiveness and safety of clinical application, a
retrospective analysis of patient data and outcome was
performed in the present study.
All values were expressed as mean ± standard deviation
(SD). Categorical variables are presented as frequency
distributions (n) and simple percentages (%). Data were
analyzed with GraphPad Prism version 7.0 for Windows
(GraphPad Software®; San Diego, CA, USA). The sample
size for the experimental design was calculated using the
free G*power 3.1-software (http://www.gpower.hhu.de).
Statistical analysis of the results was performed by
oneway analysis of variance () for repeated measures and
Tukey’s test or Student’s t test, when appropriate.
Equality of group variances was analyzed by the
BrownForsythe test. A p value less than 0.05 was considered
PCMO release pro-angiogenic and tissue recoveryassociated proteins under hypoxia
To evaluate whether transient ischemic conditions could
influence the expression pattern of PCMO for proteins
involved in angiogenesis and tissue recovery, cell lysates
(for intracellular proteins) as well as cell culture media
(for secreted proteins) of PCMO were collected 24 h
after hypoxia and normoxia, respectively. Human
proteome profiling arrays (Fig. 2a) representing 55 proteins
involved in angiogenesis and tissue regeneration were
performed with the respective samples and showed a
hypoxia-induced upregulation by more than 25% of 10/
55 (18%) proteins in culture medium and 3/55 (6%)
proteins within the PCMO cells. All investigated proteins
and their expression levels (relative optical density) are
summarized in additional Table S3 (Additional file 5:
Various proteins were upregulated in cell culture
supernatants as well as in the cell itself by more than 25%
under hypoxic conditions. In detail, the most
upregulated proteins in culture supernatants were: monocyte
chemotactic protein 1 (MCP-1; 43-fold, p < 0.0001),
macrophage inflammatory protein 1-alpha (MIP-1α;
15-fold; p < 0.0001), granulocyte-macrophage
colonystimulating factor (GM-CSF; 11-fold; p < 0.0001),
heparinbinding EGF-like growth factor (HB-EGF; 6-fold; p = 0.
0003) and pentraxin-related protein (PTX-3; 3-fold; p = 0.
0028) while within PCMO, upregulation of the
interleukins IL-1β (IL-1β; 12-fold; p < 0.0001) and IL-8 (IL-8; 1.
25-fold; p = 0.0002) dominated (Fig. 2b + c).
Supernatants from PCMO induce in vitro angiogenesis and show donor-specific potency
Tube formation assays for angiogenesis were performed
employing HUVEC that were cultivated with PCMO cell
culture supernatants (hypoxic vs. non-hypoxic
conditioned) on Matrigel-coated dishes. Endothelial tube
formation showed that supernatants of PCMO, both
hypoxic and non-hypoxic conditioned, enabled the
formation of vessel tubes in vitro (Fig. 3). Parameters of
angiogenesis (number of segments, nodes and meshes)
were evaluated and demonstrated a significant
enhancement of tube formation by hypoxic-conditioned
supernatants of donor D1 and D4 compared to PCMO
supernatants cultured from donor D2, D3 and D5 (Fig. 3).
Likewise, hypoxic-conditioned pooled samples of all five
donors generated significantly more nodes, segments and
meshes in comparison to PCMO supernatants from donor
(See figure on previous page.)
Fig. 2 Proteome profiling arrays (a) representing 55 proteins involved in angiogenesis and tissue regeneration were performed with the
respective PCMO samples. Red rectangles represent upregulated proteins in supernatants of PCMO cell culture or cells by more than 25%. The
nine most regulated proteins (b) in supernatants under hypoxia were: granulocyte-macrophage colony-stimulating factor (GM-CSF),
heparinbinding EGF-like growth factor (HB-EGF), interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1-alpha
(MIP-1α), matrix metalloproteinase (MMP-9), pentraxin-related protein (PTX-3), serpin E1 and metallopeptidase inhibitor 1 (TIMP-1). The most
regulated proteins (b) within PCMO under hypoxia were: angiogenin, coagulation factor III, interleukin-1 beta (IL-1β), interleukin-8 (IL-8), matrix
metalloproteinase (MMP-9), platelet factor 4 (PF4), metallopeptidase inhibitor 1 (TIMP-1), thrombospondin-1 and urokinase-type plasminogen
activator (uPA). The cell culture experiments showed a hypoxia-induced upregulation (c) by more than 25% of 10/55 (18%) proteins in supernatants
and 3/55 (6%) proteins within the PCMO (*p < 0.05). PCMO programmable cells of monocytic origin
D2, D3 and D5 (Fig. 3). For detailed results of the one-way
ANOVA, please refer to Table S4–6 (Additional files 6, 7,
8: Table S4–S6).
PCMO improve microcirculation in a hind limb ischemia mouse model
After inducing hind limb ischemia in mice,
microcirculation was assessed by micro-lightguide spectrophotometry
at 7-day intervals. Changes of the tissue oxygen saturation
(SO2) and blood flow were expressed as the ratio of the
ischemic (left) and non-ischemic (right) hind limb (left/right
ratio). From day 21, a significantly higher SO2 ratio was
observed in the PCMO group in contrast to the
monocytes (PCMO: 81.7% ± 4.2% vs. monocytes: 68.4% ± 3.1%;
p < 0.0001) and the placebo group (PCMO: 81.7% ± 4.2%
vs. placebo: 71.4% ± 3.4%; p < 0.0001) (Fig. 4a). Blood flow
decreased at day 14 and increased again at day 21 in all
three experimental groups (Fig. 4b). At day 21 a significant
improvment of blood flow was obserevd in the PCMO
group in contrast to the monocytes (PCMO: 74.3% ± 2.1%
vs. monocytes: 63.3% ± 3.1%; p < 0.0001) and the placebo
group (PCMO: 74.3% ± 2.1% vs. placebo: 57.5% ± 3.2%; p
< 0.0001) (Fig. 4b). At day 91, a significantly higher SO2
ratio (PCMO: 97.8% ± 4.9% vs. monocytes: 83.7% ± 4.1%;
p < 0.0001) and blood flow restoration (PCMO: 94.6% ± 4.
4% vs. monocytes: 86.4% ± 4.2%; p < 0.0001) were detected
in the PCMO group in contrast to the monocytes and the
placebo group (placebo SO2 ratio: 81.1% ± 3.9%; placebo
blood flow: 84.3% ± 4.2%; PCMO vs. placebo: p < 0.0001).
No significant differences in SO2 ratio (p = 0.163) and
blood flow recovery (p = 0.058) were observed between
the native monocytes and the placebo group during the
observation period (Fig. 4).
Further, in all three groups, toe necrosis and/or toenail
loss were observed in the ischemic leg. However, the
prevalence of necrosis was significantly lower in the
PCMO group compared to the monocytes (PCMO: 0.
227 ± 0.53 vs. monocytes: 1.045 ± 1.29; p = 0.045) and the
placebo group (PCMO: 0.227 ± 0.53 vs. placebo: 1.364 ±
1.32; p = 0.003).
PCMO prevent tissue degeneration in ischemic muscle in vivo
Chronic hind limb ischemia in mice was verified by
myoglobin staining, quantification of steatosis and the
number of muscle fibers as described before [
(Fig. 5). All three animal groups presented strong
accumulation of myoglobin and adipocytes as indication for
the chronic muscle degeneration process (Fig. 5a).
Nevertheless, the PCMO group showed significantly less
muscle atrophy (as seen in Fig. 5b) indicated by the
significantly lower decrease of muscle fibers (PCMO: 839.2
± 135 vs. monocytes: 434.2 ± 163.6; p < 0.0001) and
steatosis (PCMO: 37.6% ± 9.97% vs. monocytes: 54.65% ± 12.
97%; p = 0.001) in contrast to the monocytes and placebo
group (placebo muscle fibers: 444.6 ± 159.8; PCMO vs.
placebo: p < 0.0001; placebo steatosis: 50.5% ± 11.06%;
PCMO vs. placebo: p = 0.002). Comparision of the
monocytes and placebo group showed no significant
difference between the number of muscle fibers (p = 0.961)
and accumulation of adipocytes (p = 0.362).
Treatment with PCMO enhance neoangiogenesis in ischemic muscle in vivo
After the animal experiments, immunohistochemical
staining with endothelial antibodies against CD34 and
CD105 was performed to evaluate the effect of PCMO
on neoangiogenesis in the hind limb ischemia mouse
model (Fig. 6). Immunohistochemical examination
revealed a significantly enhanced number of CD34+
arterioles (score PCMO: 15.31 ± 4.04 vs. score monocytes: 8.
79 ± 3.78; p < 0.0001) and emerging CD105+ capillaries
(score PCMO: 12.05 ± 4.21 vs. score monocytes: 8.25 ± 3.
22; p = 0.030) in the ischemic muscle after
transplantation of PCMO in contrast to the monocytes and the
placebo group (score placebo CD34+ arterioles: 9.63 ± 3.
40; PCMO vs. placebo: p < 0.0001; score placebo CD105
+ capillaries: 7.91 ± 4.22; PCMO vs. placebo: p = 0.038).
Treatment with PCMO prevents limb amputation, induces vascular growth and improves wound healing in men
Four patients with chronic PAD and no further curative
treatment options except major amputation were treated
with injections of PCMO into the anteromedial and
posterior muscle compartment of the lower leg (Fig. 7).
No minor and/or major amputations of the lower
extremity were performed in all four patients. After 6 and
12 months no exaggerated immune response, malignant
processes or extended infection were reported. The
retrospective analysis of data is briefly summarized in
Table 1 and additional Table S2 (Additional file 4: Table
S2). After 6 months, the angiography revealed increased
growth of vascular collaterals in all patients after
treatment with PCMO (vascular collaterals prior PCMO
treatment: 8.75 ± 1.71 vs. post PCMO treatment: 13.75 ±
3.30) (Fig. 7). Data of the aftercare check-ups (at
12 months) also demonstrated improved wound healing
(wound area prior PCMO treatment: 21.67 cm2 ± 3.58
vs. post PCMO treatment: 13.27 cm2 ± 3.53 cm2) and
6minute walk test results in all patients, as presented in
During the last decade, different stem cell entities have
been investigated in experimental and small clinical
trials as a putative therapy for chronic PAD and ischemic
tissue regeneration [
]. As previously described by
our group, blood monocytes undergo a dedifferentiation
process when stimulated with a combination of
GMCSF and IL-3 and become PCMO characterized by a
unique surface phenotype consisting simultaneously of
lineage and stem cell markers [
]. Although the
mechanism by which PCMO achieve their permissive
state is still uncertain, it has been shown that the
combination of GM-CSF and IL-3 is critical in this process
and PCMO apparently share several markers of stem cell
renewal and maintenance [
]. Our group has also
provided evidence at mRNA and protein level that
critical pluripotency regulators such as octamer-binding
transcription factor 4 (OCT4), homeobox protein
NANOG and MYC are reactivated in PCMO and induce
cell plasticity . It has been assumed that PCMO
could potentially influence angiogenesis and tissue
recovery in a paracrine manner [
]. Here, we show that
PCMO improve angiogenesis and regeneration in
ischemic muscle in mice and humans and that their
properties have the potential to solve several issues in the field
of cell therapy.
The present cell culture experiments revealed high
resilience of PCMO under hypoxia implying suitability
for cell transplantion into chronic ischemic tissues. PCMO
showed a paracrine release of pro-angiogenic factors and
hypoxic-induced upregulation of proteins pointing toward
a putative ischemia-mediated secretion of angiogenetic
proteins. Both, hypoxic and non-hypoxic conditioned
supernatants of PCMO enabled vascular tube-forming in
vitro indicating a significant enhancement of vascular
tubes by hypoxic-conditioned supernatants. The hind limb
ischemia mouse model demonstrated significantly
increased neoangiogenesis, functional and morphometric
recovery of chronic ischemic muscle after transplantation
of PCMO. Healing attempts in human tend to confirm
these results without exaggerated immune response,
malignant disease or extended infection after 12 months of
One major limitation in allogeneic cell therapy is that
cell products for transplantation can only be derived
from adult stem cell populations such as bone
marrowderived stem cells or from human embryonic stem cells
generated by somatic cell nuclear transfer or from cells
with induced pluripotency [
]. Accordingly, biological,
economic, and ethical restrictions of all potential cell
sources impede the clinical feasibility. Whereas, whole
blood as a feasible source for the generation of PCMO can
be retrieved easily by a minimally invasive procedure or
acquired from waste products in blood donation [
Further, our recently described protocol enables short culture
times of PCMO with enhanced cell plasticity (4 days) .
Thus, PCMO appear as a clinical feasible and ethical
responsible source for cell therapy as well as tissue
Moreover, it is also a well-known problem in cell
therapy that transplanted cells are poorly retained in
ischemic tissues and subsequently lose their functionality
]. Interestingly, ischemia-inducible factors increased
relatively late in PCMO (Additional file 1: Figure S1)
and thus our experiments demonstrated a certain
resilience of PCMO without substantial cell damage under
hypoxia as demonstrated by marginally raised levels of
LDH at 2 h, 3 h and 4 h (Additional file 3: Figure S2). The
morphology of PCMO was slightly altered and cells
represented a more spherical and attached phenotype (as seen
in Additional file 3: Figure S2) but no significant increase
in activity of LDH was measured under hypoxia.
Despite of the well-known fact that monocytes can
partially differentiate into endothelial-like cells [
it has been hypothesized that neoangiogenesis induced
by cell transplantation may have been influenced by
paracrine secretion of pro-angiogenic effectors rather
than a direct involvement in vascular structures [
The five most upregulated proteins under hypoxia were
GM-CSF, HB-EGF, MIP-1α, PTX-3 and MCP-1, and all
of them have been strongly associated with angiogenesis
and tissue recovery [
Table 1 Patient characteristics after 12 months and vascular collaterals detected in the control angiography after 6 months
Patient Age 6-minute walk test (m) 6-minute walk test (m) Decrease of leg Ankle brachial index Increase of vascular Immunological
(y), pre-treatment post-treatment ulcer (%) post-treatment collaterals (%) events
1 57 m 97 m 323 m 42 0.7 71 None
Area of leg ulcers and numbers of vascular collaterals were expressed as percentage decrease or increase pre- and post-treatment with PCMO
GM-CSF is a cytokine with a wide range of biological
effects and its benefit for the recovery of tissue damage
has already been described [
]. Accordingly, Zhao and
colleagues demonstrated that GM-CSF accelerates
wound healing by promoting vascular endothelial
growth factor-A (VEGF-A) expression and consecutive
the proliferation of endothelial cells [
]. The mediation
of the inflammatory response by GM-CSF is also thought
to play a decisive role in skeletal muscle regeneration
following injury [
]. After muscle injury, neutrophils,
monocytes, and macrophages migrate into the damaged
tissue, and these cells are believed to tightly regulate the
proliferation of myoblasts derived from muscle stem cells
(satellite cells) that regulate muscle regeneration [
These myoblasts transiently express G-CSFR (granulocyte
colony-stimulating factor receptor) following injury and
proliferate in response to GM-CSF produced in part by
]. Moreover, GM-CSF has many
effects on differentiation of myeloid progenitors into
heterogeneous populations of monocytes and macrophages
. Pearson and colleagues hypothesized that it could
have downstream impacts on inflammatory monocytes
and macrophages that have not been fully understood yet
]. It has also been shown that GM-CSF can play a
beneficial role by switching inflammatory monocytes to
reparative type II macrophages (M2) [
]. In a mouse
model of T cell-induced colitis, GM-CSF increased the
production of IL-4, IL-10, and IL-13 and decreased the
production of interferon gamma (IFN-γ) in lamina propria
mononuclear cells. Using a modified Boyden chamber
assay, D’abritz and colleagues found that GM-CSF also
increased the ability of mononuclear cells to adhere,
migrate, and respond to microbial stimuli [
intravenous administration of GM-CSF-treated monocytes
at the onset of T cell-induced colitis significantly
ameliorated the development of disease [
]. Thus, PCMO
could have contributed to the muscle regeneration in the
present animal experiments and healing attempts by
direct stimulation of myoblasts via GM-CSF and the
enhancement of M2 macrophages. Finally, it has also been
demonstrated that GM-CSF increases vascular collateral
flow and conductance, as shown in in a short-term
administration of the cytokine in occlusive peripheral artery
disease. However, the underlying mechanisms have
remained unclear so far .
Likewise, MIP-1α and MCP-1 have been identified in
the pathways of angiogenesis and in response to vascular
inflammation by promotion of accumulation of cells
with angiogenic potential [
]. Although MIP-1α has
frequently been associated with angiogenesis, its exact
(patho)physiological function is still unclear . As
reported before, MIP-1α activates chemokine receptor
type 5 (CCR5) and c-Jun N-terminal kinase (JNK),
extracellular-signal regulated kinase (ERK), or p38 pathway
and consecutive leads to down-regulating of miR-374b
expression, which promotes VEGF-A expression and
subsequently induces human endothelial progenitor cells (EPCs)
migration and tube formation [
Further, in sites of ischemic results, local production
of MCP-1 is enhanced and monocytes are recruited via a
chemokine receptor ligand 2 (CCL2) (CCL2 = MCP-1)/
CCR2-dependent mechanism associated with a second
wave of mononuclear cell recruitment [
]. It may be
hypothesized that the recruited monocytes are quickly
converted to anti-inflammatory macrophages (M2) at
the site of injury where they participate in repair [
]. Studies of mice genetically deficient in MCP-1 or
chemokine receptor type 2 (CCR2 = MCP-1 receptor)
suggested that although not required for the early
mobilization of monocytes, the secondary wave of
monocytes recruitment and subsequent stimulation of
angiogenesis are dependent upon CCR2 signaling .
Moreover, HB-EGF has frequently been reported as a
central regulator of angiogenesis and tissue repair [
Soluble, mature HB-EGF binds to and activates
EGFreceptor, which is a critical molecular key factor to
several normal physiological processes including wound
healing, reproduction and angiogenesis [
]. In the
vascular system, expression of HB-EGF contributes to the
remodeling of vascular tissues, and decreases reactive
oxygen species (ROS) in human whole blood [
Importantly, HB-EGF has been shown to decrease ROS
production in stimulated leukocytes, one of the main
sources of ROS during tissue injury [
]. Further, it has
been demonstrated that HB-EGF restores intracellular
adenosine triphosphate (ATP) levels and preserves
cytoskeletal integrity in intestinal epithelial cells exposed to
The role of PTX-3 in angiogenesis and tissue
recovery has been controversial until today [
prototypic long pentraxin PTX-3 orchestrates the
recruitment of leukocytes, stabilizes the provisional
matrix in order to facilitate leukocyte and stem
progenitor cells trafficking, promotes swift and safe
clearance of dying cells and of autoantigens and protects
the vasculature . Accordingly, Salio and colleagues
reported that lack of PTX-3 reduced the number of
capillaries in reperfused cardiac tissue, as well as
induced a worse outcome in a study of cardiac ischemia
]. Likewise, PTX-3 has also been described to be a
factor that promotes neurogenesis and angiogenesis in
neuronal tissue [
]. Despite its beneficial effects in
angiogenesis and tissue recovery it could also be
demonstrated that high levels of soluble PTX-3 inhibits
fibroblast growth factor (FGF) 2-mediated
angiogenesis. Endothelium, when exposed to a high density of
circulating angiogenic cells (CACs), releases PTX-3
which markedly impairs the vascular regenerative
response in an autocrine manner [
]. Therefore, the
dose-dependent effect of transplanted cells and
subsequent release of angiocrine PTX-3 should be critically
assessed for the further development of cell therapies
in ischemic disease [
Collectively, the data of the hypoxia related in vitro
experiments indicates that enhanced secretion of
GMCSF, HB-EGF, MIP-1α, PTX-3 and MCP-1 by PCMO
could have contributed to the improvement of SO2
saturation, blood flow, muscle recovery and
neoangiogenesis in vivo. Remarkably, hypoxic-conditioned
supernatants of donor D1, D5, as well as pooled
supernatants from all five donors showed the
strongest increase of angiogenesis in the HUVEC tube
formation assays. These results indicate that supernatants of
donor D1 and D5 have also influenced the strong
proangiogenic effect seen in the pooled experiments and that
some donors might be better suited to achieve high
proangiogenic mononuclear cell products than others.
Possibly, interindividual immunological effects between the
donors or parameters not being represented by the
HUVEC tube formation model may have influenced these
results. Conceivably the recruitment of CACs and/or
circulating mononuclear cells by PCMO could have
contributed to the improvement of microcirculation in vivo, as
previously described [
]. Industrialized manufacturing
and the broad application of somatic cell products, require
further research on this important aspect.
In accordance with the in vitro experiments, the
animal studies demonstrated a significant
improvement of SO2 ratio after treatment with PCMO in
contrast to native monocytes and the placebo group.
Initial decrease of blood flow and velocity could be
associated with the first occurrence of tissue damage
under ischemia, whereas the significant increase of
blood flow and velocity at day 75 seem to be
associated with tissue recovery. Accordingly, the VD,
indicated by CD34+ and CD105+ vascular endothelium,
was significantly higher in mice treated with PCMO.
Neither the control group nor the monocytes group
presented significant improvements in
neoangiogenesis or blood flow restoration. Although monocytes
infiltrate dysfunctional tissues and participate in tissue
], the present results showed no
significant improvement after transplantation of native
monocytes in ischemic hind limbs. In comparison, the
upregulation of pro-angiogenic proteins in PCMO
implies a high angiogenic potency of this cell entity [
]. Remarkably, non-modulated monocytes obviously
failed to contribute effectively to neoangiogenesis and
tissue recovery indicating the importance of prior
reprogramming and preserving a prior developmental
stage of monocytes [
]. Our present findings
indicate that paracrine secretion of pro-angiogenic
factors by PCMO may have contributed relevantly to
the improved neoangiogenesis observed in mice and
men. Interestingly, the volume of muscle fibers was
significantly higher in the PCMO group and
significantly less steatosis was observed after the animal
experiments. These findings suggest a certain effect of
tissue recovery within the PCMO group and
correspond with enhanced secretion of GM-CSF and
HBEGF and its influence on extracellular matrix and
tissue regeneration [
36–38, 40, 50, 51
Despite its descriptive approach, the small series of
PCMO treatment in humans tend to confirm the results
from the in vitro and animal experiments. In all patients,
minor and major amputations of the lower extremity
could be prevented. Angiography after 6 months also
demonstrated an increased number of vascular
collaterals, thus wound healing was improved and the patients
reported a better quality of life. Aftercare at 12 months
revealed no exaggerated immune response, malignant
processes or extended infection. Nonetheless, future
randomized clinical trials should ensure a clear
differentiation from other effects and influences such as gait
training. Dose dependency, ideal type of application and
long-term side effects of transplanted PCMO are future
challenges in clinical trials, as demonstrated by the
limited experience so far and often disillusioned results
from clinical experiences with cell therapy in the past
In conclusion, PCMO are a feasible source for
proangiogenic and regenerative somatic cell therapy in
CLI. In vitro experiments revealed the pro-angiogenic
potential of PCMO and identified five significantly
upregulated proteins (GM-CSF, HB-EGF, MIP-1α,
PTX-3 and MCP-1) under hypoxic conditions. Animal
experiments as well as the first healing attempts in
humans showed improved angiogenesis, tissue
recovery and clinical outcome without adverse events. The
proteins being secreted by PCMO under hypoxia
could potentially have contributed in different ways to
improved outcome after CLI: first, by regulation of
the muscle stem cell proliferation and tissue
remodeling (GM-CSF, HB-EGF), second, by the recruitment
of circulating mononuclear cells (GM-CSF, MCP-1,
PTX-3), third, by switching inflammatory monocytes
to reparative type II (M2) macrophages (GM-CSF),
fourth, by inducing VEGF-A expression (GM-CSF,
MIP-1α). Thus, we propose that differentiated pluripotent
programmable cells of monocytic origin (PCMO) should
be further characterized for their suitability in (autologous
or allogeneic) therapies aiming at the reduction of critical
limb ischemia and also ischemia/reperfusion injury in
Additional file 1 Figure S1. Showing the evaluation of
hypoxiainduced gene expression in PCMO. (PDF 828 kb)
Additional file 2 Table S1. Presenting primers relate to
hypoxiainduced gene expression in monocytes/macrophages. (DOCX 308 kb)
Additional file 3 Figure S2. Quantification of cell damage by
measuring LDH after 1 h, 2 h, 3 h and 4 h and representative images of
PCMO cell culture under normoxia and hypoxia. (PDF 674 kb)
Additional file 4 Table S2. Presenting patient characteristics prior to
treatment with PCMO. (DOCX 18 kb)
Additional file 5 Table S3. Presenting human angiogenesis and tissue
recovery-related proteins analyzed in PCMO cell culture. (DOCX 813 kb)
Additional file 6 Table S4. Results of the tube formation assays for
angiogenesis. One-way ANOVA providing the number of segments. (XLSX
Additional file 7 Table S5. Results of the tube formation assays for
angiogenesis. One-way ANOVA providing the number of meshes. (XLSX
Additional file 8 Table S6. Results of the tube formation assays for
angiogenesis. One-way ANOVA providing the number of nodes. (XLSX
ANOVA: Analysis of variance; ATP: Adenosine triphosphate; CACs: Circulating
angiogenic cells; CCL2: Chemokine receptor ligand 2; CCR2: Chemokine
receptor type 2; CCR5: Chemokine receptor type 5; CLI: Critical limb ischemia;
ECGM: Endothelial cell growth medium; ELISA: Enzyme-linked
immunosorbent assay; EPC: Endothelial progenitor cell; ERK:
Extracellularsignal regulated kinase; EU GMP: European Union, good manufacturing
practice; FCS: Fetal calf serum; FGF: Fibroblast growth factor;
GCSFR: Granulocyte colony-stimulating factor receptor; GM-CSF:
Granulocytemacrophage colony-stimulating factor; HB-EGF: Heparin-binding EGF-like
growth factor; HE: Hematoxylin-eosin; HPF: High power fields;
HUVEC: Human umbilical vein endothelial cells; IFNγ: Interferon gamma;
IL: Interleukin; JNK: C-Jun N-terminal kinase; LDH: Lactate dehydrogenase;
M2: Prereparative type II macrophage; MCP-1: Monocyte chemotactic protein
1; M-CSF: Monocyte/macrophage colony-stimulating factor;
MIP1α: Macrophage inflammatory protein 1-alpha; p38: p38 signaling
transduction pathway; PAD: Peripheral artery disease; PBS:
Phosphatebuffered saline; PCMO: Programmable cells of monocytic origin;
PTX3: Pentraxin-related protein 3; ROS: Reactive oxygen species; VD: Vascular
density; VEGF-A: Vascular endothelial growth factor-A
This research was part of the MOIN CC-project supported by a grant from
the European Union, the German Ministry of Science and the
SchleswigHolstein’s Ministry of Science, Economy and Transport, project number:
Availability of data and materials
All data and materials are available in the manuscript.
RB, LH, KH, MA, JC and JG designed in vivo and in vitro experiments. RB, MA
and FF isolated and reprogrammed the monocytes. RB, MA, LH and KZ
performed and analyzed the cell culture experiments. RB, KH, RR, BS and JG
conducted the animal experiments. RB and KH performed and analyzed the
histological stainings and measurements of mircrocirculation during and
after the animal experiments. FF and JG conducted the healing attempts in
humans and the retrospective analysis. All authors interpreted results,
contributed to the manuscript and prepared the manuscript. AB has also
designed the in-vitro and in-vivo experiments and has also interpreted the
results, contributed to the manuscript and prepared the manuscript. All
authors read and approved the final manuscript.
Ethics approval and consent to participate
This research was in compliance with the Helsinki Declaration and approved
by the ethics committee of the University Medical Center Schleswig-Holstein,
Kiel, Germany (protocol identification: A133/04 and D411/12, respectively).
Informed written consent was obtained from all donors/participants. Human
umbilical vein endothelial cells (HUVEC) were freshly isolated from umbilical
cords and ethical approval (protocol identification: D482/14 and B315/15)
was also provided by the local ethics committee of the University Medical
Center Schleswig-Holstein, Kiel, Germany. All animal procedures were
performed in strict accordance with the Ministries of Education, Science
and Culture Guide for Care and Use of Laboratory Animals and approved by
the ethics committee of the Christian Albrecht’s University of Kiel
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
1. Dormandy J , Heeck L , Vig S. Predicting which patients will develop chronic critical leg ischemia . Semin Vasc Surg . 1999 ; 12 : 138 - 41 .
2. Norgren L , Hiatt WR , Dormandy JA , Nehler MR , Harris KA , Fowkes FGR . Tasc II Working Group. Inter-society consensus for the management of peripheral arterial disease (TASC II) . Eur J Vasc Endovasc Surg . 2007 ; 33 : S1 - S75 .
3. Gross-Fengels W , Heuser L , Vorwerk D , Helmberger T. Certification of vascular centers. Influence on quality and healthcare structures . Radiologe . 2011 ; 51 : 864 - 7 .
4. Mohammedi K , Woodward M , Hirakawa Y , Zoungas S , Colagiuri S , Hamet P , Chalmers J , ADVANCE Collaborative Group. Presentations of major peripheral arterial disease and risk of major outcomes in patients with type 2 diabetes: results from the ADVANCE-ON study . Cardiovasc Diabetol . 2016 ; 15 : 129 .
5. Wang SK , Green LA , Motaganahalli RL , Wilson MG , Fajardo A , Murphy MP . Rationale and design of the MarrowStim PAD Kit for the treatment of critical limb ischemia in subjects with severe peripheral adterial Disease (MOBILE) trial investigating autologous bone marrow cell therapy for critical limb ischemia . J Vasc Surg . 2017 ; 65 : 1850 - 7 .
6. Schweizer R , Kamat P , Schweizer D , Dennler C , Zhang S , Schnider JT , Erni D . Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner . Cytotherapy . 2014 ; 16 : 1345 - 60 .
7. Koch JM , D'Souza SS , Schwahn DJ , Dixon I , Hacker TA . Mesenchymoangioblast-derived mesenchymal stromal cells inhibit cell damage, tissue damage and improve peripheral blood flow following hindlimb ischemic injury in mice . Cytotherapy . 2016 ; 18 : 219 - 28 .
8. Xie N , Li Z , Adesanya TM , Guo W , Liu Y , Fu M , Xie X . Transplantation of placenta-derived mesenchymal stem cells enhances angiogenesis after ischemic limb injury in mice . J Cell Mol Med . 2016 ; 20 : 29 - 37 .
9. Tournois C , Pignon B , Sevestre MA , Al-Rifai R , Creuza V , Poitevin G , François C , Nguyen P. Cell therapy in critical limb ischemia: a comprehensive analysis of two cell therapy products . Cytotherapy . 2017 ; 19 : 299 - 310 .
10. Ruhnke M , Ungefroren H , Nussler A , et al. Differentiation of in vitro modified human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells . Gastroenterology . 2005 ; 128 : 1775 - 86 .
11. Dresske B , El Mokhtari NE , Ungefroren H , Ruhnke M , Plate V , Janssen D , Siebert R , Reinecke A , Simon R , Fandrich F. Multipotent cells of monocytic origin improve damaged heart function . Am J Transplant . 2006 ; 6 ( 5p1 ): 947 - 58 .
12. Compagna R , Amato B , Massa S , Amato M , Grande R , Butrico L , Franciscis ST , Serra R. Cell therapy in patients with critical limb ischemia . Stem Cells International . 2015 ; 2015 : 931420 .
13. Ai M , Yan CF , Xia FC , Zhou SL , He J , Li CP . Safety and efficacy of cellbased therapy on critical limb ischemia: A meta-analysis . Cytotherapy . 2016 ; 18 : 712 - 24 .
14. Liew A , Bhattacharya V , Shaw J , Stansby G. Cell therapy for critical limb ischemia: a meta-analysis of randomized controlled trials . Angiology . 2016 ; 67 : 444 - 55 .
15. Ungefroren H , Hyder A , Schulze M , Fawzy El-Sayed KM , Grage-Griebenow E , Nussler AK , Fändrich F . Peripheral blood monocytes as adult stem cells: molecular characterization and improvements in culture conditions to enhance stem cell features and proliferative potential . Stem Cells International . 2016 ; 2017 : 7132751 .
16. Hyder A , Ehnert S , Hinz H , Nüssler AK , Fändrich F , Ungefroren H. EGF and HB-EGF enhance the proliferation of programmable cells of monocytic origin (PCMO) through activation of MEK/ERK signaling and improve differentiation of PCMO-derived hepatocyte-like cells . Cell Commun Signal . 2012 ; 10 : 23 .
17. Huang Y , Zitta K , Bein B , Steinfath M , Albrecht M. An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells . Dis Model Mech . 2013 ; 6 : 1507 - 14 .
18. Hummitzsch L , Zitta K , Bein B , Steinfath M , Albrecht M. Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury . Exp Cell Res . 2014 ; 322 : 62 - 70 .
19. Elbarghati L , Murdoch C , Lewis CE . Effects of hypoxia on transcription factor expression in human monocytes and macrophages . Immunobiology . 2008 ; 213 : 899 - 908 .
20. Baudin B , Bruneel A , Bosselut N , Vaubourdolle M. A protocol for isolation and culture of 380 human umbilical vein endothelial cells . Nat Protoc . 2007 ; 2 : 481 - 5 .
21. Carpentier G. ImageJ contribution: angiogenesis analyzer : ImageJ News; 2012 . http://www.image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzerfor-ImageJ.html.
22. Chevalier F , Lavergne M , Negroni E , Ferratge S , Carpentier G , Gilbert-Sirieix M , Sineriz F , Uzan G , Albanese P . Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies . Stem Cell Res . 2014 ; 12 : 703 - 15 .
23. Hellingman AA , Bastiaansen AJNM , de Vries MR , Seghers L , Lijkwan MA , Löwik CW , Hammig JF , Quax PHA . Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation . Eur J Vasc Endovasc Surg . 2010 ; 40 : 796 - 803 .
24. Rother U , Kapust J , Lang W , Horch RE , Gefeller O , Meyer A . The angiosome concept evaluated on the base of micro-perfusion in CLI patients - an O2C guided study . Microcirculation . 2015 ; 22 : 737 - 43 .
25. Wilkstrom P , Lissbrant JF , Stattin P , Egevad L , Bergh A . Endoglin (CD105) is expressed on immature blood vessels and is a marker for survival in prostate cancer . Prostate . 2002 ; 51 : 268 - 75 .
26. Kukreja I , Kapoor P , Deshmukh R , Kulkarni V . VEGF and CD 34: A correlation between tumor angiogenesis and microvessel density-an immunohistochemical study . J Oral Maxillofac Pathol . 2013 ; 17 : 367 .
27. Al-Khaldi A , Al-Sabti H , Galipeau J , Lachapelle K. Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model . Ann Thorac Surg . 2003 ; 75 : 204 - 9 .
28. Badhwar A , Forbes TL , Lovell MB , Dungey AA , McCarter SD , Scott JR , DeRose G , Harris KA , Potter RF . Chronic lower extremity ischemia: a human model of ischemic tolerance . Can J Surg . 2004 ; 47 : 352 .
29. Yuan C , Yu G , Yang T , Li W , Ai Q , Deng L . Enhanced therapeutic effects on acute myocardial infarction with multiple intravenous transplantation of human cord blood mononuclear cells . Int J Cardiol . 2013 ; 168 : 2767 - 73 .
30. Fernandez Pujol B , Lucibello FC , Gehling UM , Lindemann K , Weidner N , Zuzarte ML , Adamkiewicz J , Elsässer HP , Müller R , Havemann K. Endotheliallike cells derived from human CD14 positive monocytes . Differentiation . 2000 ; 65 : 287 - 300 .
31. Schmeisser A , Garlichs CD , Zhang H , Escafi S , Graffy C , Ludwiq J , Ruth S , Werner D. Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions . Cardiovasc Res . 2001 ; 49 : 671 - 80 .
32. Sieveking DP , Ng MK . Cell therapies for therapeutic angiogenesis: back to the bench . Vasc Med . 2009 ; 14 : 153 - 66 .
33. Kumar AH , Caplice NM. Clinical potential of adult vascular progenitor cells . Arterioscler Thromb Vasc Biol . 2010 ; 30 : 1080 - 7 .
34. Mehta VB , Besner GE . HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways . Growth Factors . 2007 ; 25 : 253 - 63 .
35. Qiu C , Xie Q , Zhang D , Chen Q , Hu J , Xu L . GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK signaling . Cell Physiol Biochem . 2014 ; 33 : 784 - 95 .
36. Zhao J , Chen L , Shu B , Tang J , Zhang L , Xie J , Qi S , Xu Y. Granulocyte /macrophage colony-stimulating factor influences angiogenesis by regulating the coordinated expression of VEGF and the Ang/Tie system . PLoS One . 2014 ; 9 ( 3 ): e92691 .
37. Liao YY , Tsai HC , Chou PY , Wang SW , Chen HT , Lin YM , Chiang IP , Chang TM , Hsu SK , Chou MC , Tang CH , Fong YC . CCL3 promotes angiogenesis by dysregulation of miR-374b/VEGF-A axis in human osteosarcoma cells . Oncotarget . 2016 ; 7 : 4310 .
38. Apostolakis S , Lip GY . Shantsilla. Monocytes in heart failure: relationship to a deteriorating immune overreaction or a desperate attempt for tissue repair? Cardiovasc Res . 2010 ; 85 : 649 - 60 .
39. Vannella KM , Wynn TA . Mechanisms of organ injury and repair by macrophages . Annu Rev Physiol . 2017 ; 79 : 593 - 617 .
40. Hara M , Yuasa S , Shimoji K , Onizuka T , Hayashiji N , et al. G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation . J Exp Med . 2011 ; 208 : 715 - 27 .
41. Pearson C , Thornton EE , McKenzie B , Schaupp AL , Huskens N , et al. ILC3 GM-CSF production and mobilisation orchestrate acute intestinal inflammation . ELife . 2016 ; 5 : e10066 .
42. D'abritz J , Weinhage T , Varga G , Wirth T , Walscheid K , et al. Reprogramming of monocytes by GM-CSF contributes to regulatory immune functions during intestinal inflammation . J Immunol . 2015 ; 194 : 2424 - 38 .
43. Ito WD , Arras M , Winkler B , Scholz D , Schaper J , Schaper W. Monocyte chemotactic protein-1 increases collateral and peripheral conductance after femoral artery occlusion . Circ Res . 1997 ; 80 : 829 - 37 .
44. Apostolakis S , Vogiatzi K , Krambovitis E , Spandidos DA . IL-1 cytokines in cardi- ovascular disease: diagnostic, prognostic and therapeutic implications . Cardiovasc Hematol Agents Med Chem . 2008 ; 6 : 150 - 8 .
45. Salcedo R , Ponce ML , Young HA , Wasserman K , Ward JM , Kleinman HK , Oppenheim JJ , Murphy WJ . Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression . Blood . 2000 ; 96 : 34 - 40 .
46. Tsirakis G , Roussou P , Pappa CA , Kolovou A , Vasilokonstantaki C , Miminas J , Kyriakaki S , Alegakis A , Alexandrakis M. Increased serum levels of MIP-1alpha correlate with bone disease and angiogenic cytokines in patients with multiple myeloma . Med Oncol . 2014 ; 31 : 778 .
47. Jiang HR , Milovanovic M , Allan D , Niedbala W , Besnard AG , et al. IL -33 attenuates EAE by suppressing IL-17 and IFN-γ production and inducing alternatively activated macrophages . Eur J Immunol . 2012 ; 42 : 1804 - 14 .
48. Shechter R , Miller O , Yovel G , Rosenzweig N , London A , et al. Recruitment of beneficial M2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus . Immunity . 2013 ; 38 : 555 - 69 .
49. Taylor SR , Markesbery MG , Harding PA . Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and proteolytic processing by a disintegrin and metalloproteinases (ADAM): a regulator of several pathways . Semin Cell Dev Biol . 2014 ; 28 : 22 - 30 . https://doi.org/10.1016/j.semcdb. 2014 . 03 .004.
50. Zhang H , Sunnarborg SW , McNaughton KK , Johns TG , Lee DC , Faber JE . Heparin-binding epidermal growth factor-like growth factor signaling in flow-induced arterial remodeling . Circ Res . 2008 ; 102 : 1275 - 85 .
51. Kuhn MA , Xia G , Mehta VB , Glenn S , Michalsky MP , Besner GE . Heparinbinding EGF-like growth factor (HB-EGF) decreases oxygen free radical production in vitro and in vivo . Antioxid Redox Signal . 2002 ; 4 : 639 - 46 .
52. Erreni M , Manfredi AA , Garlanda C , Mantovani A , Rovere-Querini P. The long pentraxin PTX3: A prototypical sensor of tissue injury and a regulator of homeostasis . Immunol Rev . 2017 ; 280 : 112 - 5 .
53. Rodriguez-Grande B , Swana M , Nguyen L , Englezou P , Maysami S , Allan SM , et al. The acute-phase protein PTX3 is an essential mediator of glial scar formation and resolution of brain edema after ischemic injury . J Cereb Blood Flow Metab . 2014 ; 34 : 480 - 8 .
54. Salio M , Chimenti S , De Angelis N , Molla F , Maina V , Nebuloni M , et al. Cardioprotective function of the long pentraxin PTX3 in acute myocardial infarction . Circulation . 2008 ; 117 : 1055 - 64 .
55. Rodriguez-Grande B , Varghese L , Molina-Holgado F , Rajkovic O , Garlanda C , Denes A , Pinteaux E. Pentraxin 3 mediates neurogenesis and angiogenesis after cerebral ischaemia . J Neuroinflammation . 2015 ; 12 : 15 .
56. O 'Neill CL , Guduric-Fuchs J , Chambers SE , O'Doherty M , Bottazzi B , Stitt AW , Medina RJ . Endothelial cell-derived pentraxin 3 limits the vasoreparative therapeutic potential of circulating angiogenic cells . Cardiovasc Res . 2016 ; 112 : 677 - 88 .
57. Odent G , Preda MB , Radu E , Rosca AM , Tutuianu R , Mitroi DN , Simionescu M , Burlacu A . Combinatorial approach for improving the outcome of angiogenic therapy in ischemic tissues . Biomaterials . 2015 ; 60 : 72 - 81 .