Cell. Mol. Life Sci.

DOI 10.1007/s00018-017-2665-z Cellular and Molecular Life Sciences

ORIGINAL ARTICLE

Mesenchymal stem cells from preterm to term newborns undergo

a significant switch from anaerobic glycolysis to the oxidative
phosphorylation
Silvia Ravera1   · Marina Podestà1 · Federica Sabatini1 · Chiara Fresia2 ·
Marta Columbaro3 · Silvia Bruno4 · Ezio Fulcheri5 · Luca Antonio Ramenghi6 ·
Francesco Frassoni1 

Received: 20 March 2017 / Revised: 4 September 2017 / Accepted: 25 September 2017

© Springer International Publishing AG 2017

Abstract  We evaluated the energy metabolism of human distribution inside the cell; in fact, CLUH silencing in term
mesenchymal stem cells (MSC) isolated from umbilical cord MSC determined a metabolic fingerprint similar to that of
(UC) of preterm (< 37 weeks of gestational age) and term preterm MSC. Our study discloses novel information on the
(≥ 37 weeks of gestational age) newborns, using MSC from production of energy and mitochondrial organization and
adult bone marrow as control. A metabolic switch has been function, during the passage from fetal to adult life, provid-
observed around the 34th week of gestational age from a ing useful information for the management of preterm birth.
prevalently anaerobic glycolysis to the oxidative phosphoryl-
ation. This metabolic change is associated with the organiza- Keywords  CLUH · Endothelial cells · Energy
tion of mitochondria reticulum: preterm MSCs presented a metabolism · Mesenchymal stem cells · OXPHOS ·
scarcely organized mitochondrial reticulum and low expres- Preterm newborns · Term newborns
sion of proteins involved in the mitochondrial fission/fusion,
compared to term MSCs. These changes seem governed
by the expression of CLUH, a cytosolic messenger RNA- Introduction
binding protein involved in the mitochondria biogenesis and
Human mesenchymal stem cells (MSCs) are somatic cells
that display a great proliferation capacity, multi-lineage dif-
Electronic supplementary material  The online version of this ferentiation and an extensive self-renewal ability [1]. At first,
article (doi:10.1007/s00018-017-2665-z) contains supplementary MSCs have been isolated from adult tissues, such as bone
material, which is available to authorized users.
marrow (BM) or adipose tissue, but these cells seem to lose
* Silvia Ravera their proliferative potential with aging [1]. Recently, also the
silvia.ravera@gmail.com umbilical cord (UC) has been considered as a great source of
1
MSC, because it contains a considerable number of MSCs
Stem Cell Laboratory and Cell Therapy Center, IRCCS
with properties similar to those of MSCs from bone marrow
Istituto Giannina Gaslini, 16147 Genoa, Italy
2
and adipose tissue [2], characterized by higher proliferative
Section of Biochemistry, Department of Experimental
rates [3].
Medicine, University of Genoa, 16132 Genoa, Italy
3
There is an increasing interest in understanding rela-
SC Laboratory of Musculoskeletal Cell Biology, IRCCS
tionships between energy metabolism, differentiation, and
Rizzoli Orthopedic Institute, 40136 Bologna, Italy
4
proliferative potential. It is known that murine embryonic
Section of Human Anatomy, Department of Experimental
stem cells (mESCs) express a high glycolytic flux for their
Medicine, University of Genoa, 16132 Genoa, Italy
5
energy production probably to minimize reactive oxygen
Laboratory Medicine and Diagnostic Services, Division
species (ROS) production [4–6]. Moreover, the anaerobic
of Perinatal Pathology, Department of Translational
Research, IRCCS Istituto Giannina Gaslini, 16147 Genoa, metabolism shifts to aerobic metabolism only during the
Italy differentiation in somatic cells [7, 8], suggesting a mitochon-
6
Neonatal Intensive Care Unit, IRCCS Istituto Giannina drial role in the bioenergetic functions associated with the
Gaslini, 16147 Genoa, Italy differentiation of stem cells [9, 10]. For example, Shum et al.

13
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have observed that BM-MSCs displayed a high OXPHOS
metabolism during osteogenic differentiation, albeit main-
taining the glycolysis rate similar to that of undifferentiated
cells [11]. Other authors have observed that adult stem cells
remain in a prevalent quiescent state, down-regulating meta-
bolic activity and relying mainly on glycolysis. However,
glycolysis is not, per se, associated with low pace of prolif-
eration since induced pluripotent stem cells (iPS) may pro-
liferate indefinitely while maintaining high glycolytic rate
and globular immature mitochondria [12].
Although mitochondria are considered the powerhouse of
the cells, their activity is not limited to the energy produc-
tion, but they are also involved in the regulation of cell cycle
and proliferation, through ROS production and the apoptosis
processes [13].
Recently, we have described that only the MSC-derived
exosomes from full-term newborns [≥ 37 weeks of gesta-
tional age (GA)] are able to conduct a complete oxidative
phosphorylation (OXPHOS) activity, producing ATP, while
exosomes from preterm newborns (< 37 weeks of GA) con-
sumed oxygen, but did not produce ATP [14]. These data
anticipated that the MSCs undergo a “metabolic shift” from
anaerobic to aerobic, during gestational development.
After those initial observations, in this work, we have
evaluated the asset of energy metabolism and the function
of mitochondria in the development of UC-MSCs isolated
from preterm and term newborns.
Materials and methods
Cell cultures
After written informed consent, umbilical cords from full-
term (gestational age (GA) ≥ 37 weeks, n = 89, term) and
preterm (GA 26, 28, 34 and 36 weeks, n = 88 for each age)
babies were collected following elective cesarean section
[15]. Study was approved by the Institutional Review Board
of the IRCCS G. Gaslini, Genoa, Italy (no. 164, 22nd July
2013).
The MSCs were isolated from umbilical cord (UC) using
a non-enzymatic digestion protocol [16]. Briefly, the adher-
ent cells growing from small pieces of cord were collected
after 14 days of culture and re-plated at low density (500/
cm2), using the same culture conditions until confluence,
in a 75-cm2 culture flask. Finally, cells were harvested,
counted, and identified by flow cytometry using standard
mesenchymal markers, at P2 [17]. Only samples presenting
a purity ≥ 95% have been evaluated for the energetic status.
MSCs isolated from adult bone marrow [18] were used as
positive control.
All cells were cultured in Dulbecco’s modified eagle
medium (DMEM), low glucose medium (Gibco Invitrogen,
13
S. Ravera et al.
Carlbad, CA, USA) containing penicillin/streptomycin,
Glutamine (Gibco Invitrogen, Carlbad, CA, USA) heparin
(PharmaTex Italia, Milan, Italy), and 5% platelet lysate at
37 °C in 5% carbon dioxide (­ CO2) and in 21% O­ 2 (normoxic
condition). For hypoxic condition, UC-MSCs were incu-
bated for 48 h in a humidified hypoxic chamber (Binder,
Tuttlingen, Germany) at 5% C ­ O2 level and 1% O­ 2 balanced
with ­N2.
Endothelial cells were obtained from cord blood samples
of premature and full-term infants as described elsewhere
[19]. The cells were cultured in endothelial cell growth
medium EGM-2 Bullet Kit (Lonza-Euroclone) at 37 °C in
5% ­CO2 and used in passage numbers 2–4.
Proliferative potential
Adherent cells growing from small pieces of cords were har-
vested when they became confluent in a 100-mm Petri dish
and were counted (p1MSC).
Adherent cells growing from 3.8 × 104 p1MSC were har-
vested and counted when they were confluent in a 75-cm2
tissue culture flask (p2MSC).
MSC immunophenotyping
Immunophenotyping of the expanded term or preterm UC-
derived MSCs was done using flow cytometry, at passage
2 [14]. After detachment using 0.05% trypsin/EDTA, cells
were washed, pelleted, and stained with fluorescein isotiocy-
anate-, phycoerythrin-, allophycocyanin- or VioBlue-conju-
gated monoclonal antibodies specific for CD14 (345784, BD
Biosciences), CD31 (560984, BDBiosciences), CD34 (130-
095-393, MiltenyiBiotec), CD45, (560976, BDBiosciences),
CD73 (130-097-947, MiltenyiBiotec), CD90 (130-095-400,
MiltenyiBiotec), CD105 (130-094-941, MiltenyiBiotec),
CD166 (559263, BDpharmigen) and SSEA-4 (130-098-
369, MiltenyiBiotec). Nonspecific staining was determined
using isotype-matched nonreactive mouse IgGs and 7-AAD
(EBioScience). 7-AAD (EBioScience) was used to evalu-
ate cell viability. After washing, at least 5000 events were
acquired on MACSQuant Analyzer 10 (MiltenyiBiotec) and
the multicolor analysis was performed using FlowJo 10.0.6
software (Tree Star, Ashland, OR, USA).
Intracellular staining for the detection of pluripotency
gene was performed on UC-MSC as previously described
[19]. Cells were fixed/permeabilized using Fix/Perm buffer
kit (BD Biosciences) according to manufacturer’s indica-
tions. The following PE-conjugated antibodies were used:
Nestin, (Pharmigen), OCT3/4 (EBioScience), and Nanog
(BD Pharmigen). Samples were then acquired on MACS-
Quant and data were analyzed by FlowJo.
Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic…
Small interfering RNA and transfection
Predesigned siRNAs against human CLUH (5′-CCA​CCA​
GCU ​ G GA​ C CA​ C GU ​ C UU ​ UAA ​ A -3′) and nonspecific
siRNA (named scramble) for control were obtained from
Life Technologies. Transfection of siRNA was performed
as described previously [20]. Briefly, MSCs were grown
in either six-well or eight-well Lab-Tek chamber slide at
5 × 1­ 04 cells/cm2 in DMEM with 5% platelet lysate. At
70–80% confluence, cells were incubated with siRNA
transfection solution at 37  °C 5% C ­ O 2. After 24  h of
down-regulation, metabolic assays and confocal micros-
copy analyses were performed.
Evaluation of ATP/AMP ratio
To calculate the ATP/AMP ratio, the intracellular concentra-
tion of ATP and AMP was evaluated spectrophotometrically
[21]. For each assay, 20 µg of total protein was used. ATP
was assayed, following NADP reduction at 340 nm, in a
solution containing: 50 mM Tris–HCl, pH 8.0, 1 mM NADP,
10 mM ­MgCl2, and 5 mM glucose in 1 ml of final volume.
Samples were analyzed before and after the addition of 4 µg
of purified hexokinase/glucose-6-phosphate dehydrogenase.
AMP was assayed following the NADH oxidation at
340 nm. The medium contained 100 mM Tris–HCl, pH 8.0,
75 mM KCl, 5 mM ­MgCl2, 0.2 mM ATP, 0.5 mM phos-
phoenolpyruvate, 0.2 mM NADH, 10 IU adenylate kinase,
25 IU pyruvate kinase, and 15 IU of lactate dehydrogenase.
Oxygen consumption measurements
O2 consumption was evaluated using 100,000 preterm or
term UC-MSC or endothelial cells, permeabilized with
0.03% digitonin by a thermostatically controlled oxy-
graph apparatus equipped with an amperometric electrode
(Unisense-Microrespiration, Unisense A/S, Denmark).
Before each experiment, the electrode was equilibrated
with the respiration medium containing: 137 mM NaCl,
5 mM KCl, 0.7 mM K ­ H2PO4, 25 mM Tris–HCl, pH 7.4, and
25 mg/ml ampicillin. The addition of the respiring substrates
was performed with a Hamilton syringe, in the following
order: 10 mM pyruvate plus 5 mM malate, 40 µM rotenone,
20 mM succinate, and 50 µM antimycin A. Pyruvate and
malate were used to stimulate the pathway composed by
Complexes I, III and IV, while succinate was used for the
pathway formed by Complexes II, III and IV. To observe the
ADP-stimulated respiration rates, 0.08 mM ADP was added
after the addition of pyruvate and malate or succinate.
The respiratory rates were expressed as nmol O/min/106
cells [22].
Fo–F1 ATP synthase activity assay
To evaluate the Fo–F1 ATP synthase activity, 100,000
preterm or term UC-MSC or endothelial cells were incu-
bated for 10 min at 37 °C in a medium containing: 10 mM
Tris–HCl, pH 7.4, 100 mM KCl, 5 mM K ­ H2PO4, 1 mM
EGTA, 2.5 mM EDTA, and 5 mM ­MgCl2, 0.6 mM oua-
bain and 25 mg/ml ampicillin. Afterward, ATP synthesis
was induced by the addition of 10 mM pyruvate plus 5 mM
malate or 20 mM succinate, to stimulate the pathways com-
posed by Complexes I, III, and IV pathway or Complexes
II, III, and IV, respectively. The reaction was monitored for
2 min, every 30 s, in a luminometer ­(GloMax® 20/20n Lumi-
nometer, Promega Italia, Milano, Italy), by the luciferin/
luciferase chemiluminescent method. ATP standard solu-
tions between 1­ 0−8 and 1­ 0−5 M was employed (luciferin/
luciferase ATP bioluminescence assay kit CLSII, Roche,
Basel, Switzerland). Data were expressed as nmol ATP pro-
duced/min/106 cells [23].
The oxidative phosphorylation efficiency (P/O ratio)
was calculated as the ratio between the concentration of the
produced ATP and the amount of consumed oxygen in the
presence of respiring substrate and ADP. When the oxygen
consumption is completely devoted to the energy produc-
tion, the P/O ratio should be around 2.5 and 1.5 after pyru-
vate plus malate or succinate addition, respectively [24].
Evaluation of glycolysis flux
To assay the glycolytic flux, the activities of hexokinase,
phosphofructokinase, pyruvate kinase, and lactate dehy-
drogenase were measured at room temperature on 20 µg of
preterm or term UC-MSCs or endothelial cells total pro-
tein. Enzymatic activity was expressed as mU/mg of total
protein (nmol/min/mg of protein). NADH molar extinction
coefficient was considered 6.22 mM-1 ­cm-1, at 340 nm. The
reaction mixtures used for the determination of each enzyme
activity were the following [25]:
Hexokinase (EC 2.7.1.1): 100  mM Tris–HCl pH 7.4,
5 mM ­MgCl2, 200 mM glucose, 1 mM ATP, 0.91 mM NADP,
and 0.55 IU/ml of glucose 6-phosphate dehydrogenase.
Phosphofructokinase (EC 2.7.1.11): 100 mM Tris–HCl,
pH 7.4, 2 mM M ­ gCl2, 5 Mm KCl, 2 mM fructose 6-phos-
phate, 1 mM ATP, 0.5 mM phosphoenolpyruvate, 0.2 mM
NADH, and 2  IU/ml of pyruvate kinase and lactate
dehydrogenase.
Pyruvate kinase (EC 2.7.1.40): 100 mM Tris–HCl, pH
7.6, 2.5 mM ­MgCl2, 10 mM KCl, 0.6 mM phosphoenolpyru-
vate, 0.2 mM NADH, 5 mM ADP, and 1 IU/ml of lactate
dehydrogenase.
Lactate dehydrogenase (EC 1.1.1.27): 100 mM Tris–HCl
pH 7.4, 0.2 mM NADH, and 5 mM pyruvate.
13

The lactate concentration was measured in the growth
medium, following the reduction of ­NAD+. The assay mix-
­ AD+, and
ture contained: 100 mM Tris–HCl pH 7.4, 5 mM N
4 IU of lactate dehydrogenase.
Electrophoretic separation and semiquantitative WB
Denaturing electrophoresis (sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) was performed
following a Laemmli protocol with minor modifications in
a Mini-PROTEAN III (BioRad, Hercules, CA, USA) appa-
ratus. Separating gel was a gradient gel from 6 to 20% w/v
acrylamide. For each sample, 20 µg of total protein was
loaded. Run was performed at 4 °C, at 70 mA for each gel,
for 120–150 min.
Electrophoretically separated samples were transferred
onto nitrocellulose (NC) membranes by electroblotting, at
400 mA for 1 h, at 4 °C. NC was incubated with the spe-
cific Abs (overnight at 4 °C) diluted in PBS plus 0.15%
tween (PBSt): anti-MTCO2 (subunit II of complex IV,
ab110258, Abcam); anti-ATP5B (β subunit of ATP syn-
thase, HPA001528, Sigma Aldrich); anti-OPA1 (ab42364,
Abcam); anti-DRP1 (ab54038, Abcam); anti-eIF3X (CLUH,
A301-764A, Bethyl Lab); SOD-2 (superoxide dismutase iso-
form 2, sc-18503, Santa Cruz Biotechnology) and anti-β-
actin antibody (sc-47778, Santa Cruz Biotechnology). Sec-
ondary HPR-conjugated Abs were diluted in PBS 1:10,000.
Bands were visualized by chemiluminescence with Immuno-
Star WesternC kit (BioRad Lab, Hercules, CA) and images
acquired by means of the molecular imager ChemiDoc
XRS + System (Bio-Rad). Quantitative densitometry was
performed using the ChemiDoc XRS + System software and
data expressed as relative optical density (R.O.D.).
Transmission electron microscopy
For this analysis, UC-MSCs pellets from preterm and term
newborns were employed. Cell pellets were fixed with 2.5%
glutaraldehyde/0.1 M cacodylate buffer at pH 7.6 for 1 h
at room temperature. After post-fixation with 1% ­OsO4 in
cacodylate buffer for 1 h, pellets were dehydrated in an etha-
nol series and embedded in Epon resin. Ultrathin sections
stained with uranyl-acetate and lead citrate were observed
with a Jeol Jem-1011 transmission electron microscope. 200
mitochondria were examined for each sample.
Determination of lipid peroxidation: malondialdehyde
assay
To assess the lipid peroxidation in preterm and term UC-
MSCs or endothelial cells, malondialdehyde (MDA) levels
were assayed, using the thiobarbituric acid reactive sub-
stances (TBARS) assay, with minor modifications [26].
13
S. Ravera et al.
This method is based on the reaction of MDA, a breakdown
product of lipid peroxides, with thiobarbituric acid TBA.
To evaluate the basal concentration of MDA, 50 μg of total
protein dissolved in 300 μl of milliQ water was added to
600 μl of TBARS solution, containing 15% trichloroacetic
acid in 0.25 N HCl and 26 mM TBA.
The mix was incubated for 40 min at 100 °C, centrifuged
at 14,000 rpm for 2 min, and the supernatant was analyzed
spectrophotometrically, at 532 nm.
Different MDA concentrations (0.75, 1, and 2 μM) were
used to obtain a standard curve.
Catalase activity assay
Catalase activity was evaluated following the peroxidation
of ­H2O2, at 240 nm. The assay medium containing: 100 mM
Tris–HCl, pH 7.4, and 2 mM H ­ 2O2. For each assay, 20 µg of
total protein was employed.
Confocal microscopy
Cells were washed with growth medium and incubated with
200 nM MitoTracker-633 probe (Life Technologies Ltd,
Monza, MB, Italy), for 20 min at room temperature. After
the incubation, sample was washed with PBS and imme-
diately analyzed using a laser scanning spectral confocal
microscope TCS SP2 AOBS (Leica, Heidelberg, Germany),
equipped with Argon ion, He–Ne 543  nm, and He–Ne
633 nm lasers. Images were acquired through a HCX PL
APO CS 40 ×/1.25 oil UV objective and processed with
Leica. Images were acquired as single transcellular optical
sections.
Gene expression analysis
Total RNA was isolated from 0.5 to 1 × ­105 term or pre-
term UC-MSC, using the RNeasyPlus Mini kit (QIAGEN),
according to the manufacturer’s protocol. Amount and purity
of RNA were assessed using a NanoDrop spectrophotometer
(Nanodrop Technologies) and 1 µg of total RNA was used
to synthesize cDNA by reverse transcription, using iScript
Reverse Transcriptase (BioRad). SYBR Green q-PCR was
performed on an iQ5 (Bio-Rad). Results were normalized
into two reference genes, β-actin and TATA-box binding
protein, selected by NormFinder for their highest stability
among a pool of common housekeeping genes [27]. Primers
were designed by NCBI/primer-BLAST primer designing
tool. Statistical analysis of the results was performed using
the 2ΔCt method [28], and results were expressed relative to
term UC-MSC expression values. CLUH primers sequences
were TACA ​ TCA​ TGG ​ GCG​ ACT ​ ACG​ C (forward primer) and
GGC​CAG​GTG​CAT​GTA​TTC​CT (reverse primer).
Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic…
Statistical analysis
GraphPad Prism 4.03 for Windows (GraphPad Software, La
Jolla, CA, USA) was used to carry out statistical analysis.
Two-way repeated-measures ANOVA was used to evaluate
the changes among term and preterm samples; the Bonfer-
roni post hoc test was then used to evaluate the difference
between treatments at specific time points.
Results
Characterization of preterm and term UC‑MSCs
UC-MSCs derived from preterm and term newborns were
characterized by flow cytometry at passage 2. Both samples
met the standard criteria [17]. In fact, the majority of col-
lected preterm and term UC-MSCs were highly positive for
the main MSCs markers CD73, CD90, and CD105, as well
as of CD166, but negative for the typical hematopoietic and
endothelial markers, such as CD14, CD31, CD34, and CD45
(Figure 1 Supplementary, panel a). Furthermore, the expres-
sion of stemness markers, such as Nestin, Oct3/4, Nanog,
and SSEA-4, did not differ in UC-MSC from preterm and
term samples (Figure 1 Supplementary, panel b).
In addition, as reported in Fig. 1, preterm cord samples
(n = 89) grew a higher number of MSC both in the first and
in the second generation compared to term cords (n = 88)
(p = 0.01 and p = 0.003, respectively), although both sam-
ples maintained a high proliferation rate.
The identity of endothelial cells from preterm and term
newborns was previously reported [19].
Fig. 1  Proliferation rate of preterm and term UC-MSCs. Graph
shows the proliferation rate of preterm (gray columns) and term
(white columns) UC-MSCs at first or the second passage in culture
(P1 and P2, respectively). Preterm cord samples (no. 89) grew a
higher number of MSCs both at the first than at the second generation
compared to term cords (no. 88) (p  =  0.01 and p  =  0.003, respec-
tively). Asterisk indicates a p  <  0.003 between preterm and term at
P1. Double asterisk indicates a p < 0.001 between preterm and term
at P2
Energy status of preterm and term UC‑MSCs
The intracellular level of ATP appeared similar in each
sample (Fig. 2, panel a), while the AMP concentration
decreased with GA progressing (Fig.  2, panel b). This
determined an increment of the ATP/AMP ratio propor-
tionally with the GA of the sample, reaching a maximum
in term UC-MSCs and BM-MSCs, used as positive control
(Fig. 2, panel c).
Energy metabolism in preterm and term UC‑MSCs
The difference observed in the ATP/AMP ratio may depend
by the pathways that UC-MSCs employ for the energy pro-
duction, thus, the activity of the oxidative phosphorylation
(OXPHOS; in term of oxygen consumption and ATP syn-
thesis), as well as the activity of some glycolytic enzymes
was investigated. Data show that the oxygen consumption
and the ATP synthesis (Fig. 3, panels a, b, respectively)
were very low in the early preterm stage (26–28 weeks) and
increased concurrently with the GA.
In addition, when early preterm UC-MSCs were stimu-
lated with pyruvate plus malate in comparison with succi-
nate stimulation, they displayed a lower P/O ratio compared
to samples with higher GA, suggesting that the OXPHOS
machinery displays low efficiency (Fig. 3, panel c).
The increment of the OXPHOS activity during fetal life
was confirmed by the expression of COXII, a complex IV
subunit codified by mitochondrial DNA and by the expres-
sion of β subunit of Fo–F1 ATP synthase, codified by
nuclear DNA (Fig. 3, panels d–f). By contrast, the activity
of hexokinase (HK), phosphofructokinase (PFK), pyruvate
kinase (PK) and lactate dehydrogenase (LDH) as well as
the lactate release were very high in preterm UC-MSCs and
decrease proportionally with their maturation (Fig. 3, panels
g–k). Moreover, as reported in Fig. 3, panel l, the LDH activ-
ity is inversely proportional to the oxygen consumption rate.
Oxidative stress production
The mitochondrial activity is always associated with the oxi-
dative stress production. Therefore, we evaluated the MDA
level, a marker of lipid peroxidation, the catalase activity,
and SOD2 expression. As reported in Fig. 4, panel a, MDA
concentration was low in preterm UC-MSCs until the 28th
week of GA, increased around the 34th week of GA, reach-
ing a maximum around the term stage. On the other side,
both catalase activity (Fig. 4, panel b) and SOD2 expres-
sion (Fig. 4, panels c, d) increased concurrently with the
UC-MSC maturation, suggesting an improvement of the
endogenous antioxidant defenses.
13

Fig. 2  Energetic status of preterm and term UC-MSCs. a shows the
intracellular concentration of ATP in preterm (26–36 weeks of GA),
term (39 weeks of GA), and BM-MSCs. The values appear similar
in all samples, without significant differences. b Reports the intracel-
lular concentration of AMP in preterm (26–36 weeks of GA), term
(39 weeks of GA), and BM-MSCs. The values increase proportional
to the age of the samples. The same trend was observed for the ATP/
AMP ratio, showed in c. All data are expressed as mean  ±  SD and
Mitochondria morphology, organization, and biogenesis
To understand whether the limited aerobic metabolism in
preterm UC-MSCs depends on either a lower mitochondria
density or alterations of mitochondria structures, TEM and
confocal microscopy analysis were performed. (Fig. 5, pan-
els a, b). Preterm as well as term and bone marrow MSC
showed comparable number of well-developed mitochon-
dria, with rounded or elongated shape, characterized by
normally structured cristae and dense matrix. However, the
mitotracker staining showed that while in term and BM-
MSCs mitochondria were organized, as expected, in a reticu-
lum (Fig. 5, panel c), in preterm samples, they were scattered
within the cytoplasm.
We have further evaluated, by western blot analyses, the
expression of OPA1, a protein belonging to the inner mem-
brane involved in the fusion, and DRP1, a protein expressed
in the outer mitochondria membrane involved in the fission.
Both proteins were virtually absent in preterm UC-MSCs,
while they were expressed in term and BM-MSCs (Fig. 5,
13
S. Ravera et al.
are representative of at least four experiments. There is not sig-
nificance different among term UC-MSCs (39 weeks of GA) and
BM-MSCs. Double asterisk indicates a significant difference for
p  <  0.001 between preterm (26–34 weeks of GA) and term UC-
MSCs (39 weeks of GA). Asterisk indicates a significant difference
for p < 0.005 between preterm (26–34 weeks of GA) and term UC-
MSCs (39 weeks of GA)
panel d). Data are confirmed by the densitometric analysis
reported in Fig. 5, panel e.
Role of CLUH in mitochondrial physiology
To understand whether the changes in mitochondria endo-
plasmic reticulum organization correlated with the expres-
sion of CLUH, a ribosomal factor involved in the mito-
chondria biogenesis and distribution [20, 29], RT-PCR, and
western blot for CLUH was performed. In preterm samples,
CLUH mRNA expression and protein signal were low but
increased concurrently with the GA (Fig.  6, panel a–c,
respectively).
In addition, by silencing CLUH in term UC-MSCs,
a shift from aerobic to anaerobic metabolism was docu-
mented with a significant decrement of OXPHOS pro-
teins and OPA1 expression compared to the control and
the scramble sample (Fig. 7, panels a–e). The decrease
of OXPHOS proteins expression accomplished an impair-
ment of ATP/AMP ratio, oxygen consumption, ATP
Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic…
Fig. 3  Energy metabolism in preterm and term UC-MSCs. The
mitochondrial oxygen consumption (a) and the ATP synthesis from
Fo–F1 ATP synthase (b) were assayed to evaluate the mitochondrial
oxidative metabolism in preterm (26, 28, 34 and 36 weeks of GA),
term (39 weeks of GA) and BM-MSCs. To stimulate the pathway
composed by Complexes I, III, and IV, pyruvate    plus  malate (Pyr/
Mal; gray columns) was employed; while succinate (white columns)
was used to stimulate the pathway formed by complexes II, III, and
IV. In both cases, the mitochondrial function is very low in the early
preterm samples (26–28 weeks of GA), increasing proportionally to
the age with a maximum in term (39 weeks of GA) and BM MSC.
All data are expressed as mean  ±  SD and are representative of at
least five experiments. There is not significance different between
term (39 weeks of GA) and BM MSC. Double asterisk indicates a
significant difference for p < 0.001 with respect the value of term (39
weeks of GA) and BM MSC. c Reports the P/O value after the addi-
tion of pyruvate    plus  malate or succinate. This parameter indicates
the efficiency of the OXPHOS machinery and must be around 2.5 for
pyruvate  plus malate induction and 1.5 for the succinate addition. All
data are expressed as mean  ±  SD and are representative of at least
five experiments. d shows the WB analysis against COXII, a subu-
synthesis, MDA production, and catalase activity (Fig. 7,
panel f–l). Finally, the mitochondrial reticulum of term
UC-MSCs appeared less organized with respect to the
controls (Fig. 7, panel m). By contrast, the activities of
nit of complex IV, and the β subunit of ATP synthase. The loaded
samples were: preterm UC-MSCs (28, 32 and 34 weeks of GA), term
UC-MSCs (39 weeks of GA), and BM-MSCs used as positive con-
trol. The densitometric analysis of the WB signals is shown in e and
f, respectively. d–f are representative of at least four experiments and
double asterisk indicates a significant difference for p  <  0.001 with
respect to the value of term (39 weeks of GA) and BM MSC. To eval-
uate the glycolytic flux and the anaerobic metabolism in preterm (26–
36 weeks of GA), term (39 weeks of GA), and BM-MSCs, the activi-
ties of hexokinase, (HK, g), phosphofructokinase (PFK, h), pyruvate
kinase (PK, i) lactate dehydrogenase (LDH, j) and the lactate release
(k) were assayed. In all cases, the activity of these enzymes and the
lactate concentration decrease proportionally with the age of the sam-
ple, reaching a minimum value in term (39 weeks of GA) and BM-
MSCs. The correlation between the oxygen consumption rate (OCR)
and the LDH activity is shown in l. Data are expressed as mean ± SD
and are representative of at least five experiments. There is not sig-
nificance different between term (39 weeks of GA) and BM MSC.
Double asterisk indicates a significant difference for p < 0.001 among
preterm UC-MSCs with respect to the value of term UC-MSCs (39
weeks of GA) and BM-MSCs
glycolytic enzymes (HK, PFK, PK, and LDH; Fig. 7, panel
i) as well as the lactate production (Fig. 7, panel j) were
increased in the CLUH silencing sample in comparison
with the controls.
13

Fig. 4  Oxidative stress production in preterm and term UC-MSCs.
a shows the concentration of MDA, a lipid peroxidation marker, in
preterm (26–36 weeks of GA), term (39 weeks of GA), and BM-
MSCs. b shows the catalase activity in preterm (26–36 weeks of
GA), term (39 weeks of GA), and BM-MSCs. In these panels, data
are expressed as mean  ±  SD and are representative of at least five
experiments. There is not significance different among term (39
weeks of GA) and BM MSC. Double asterisk indicates a significant
difference for p < 0.001 among preterm UC-MSCs with respect to the
Effect of hypoxia on the metabolism of preterm
and term UC‑MSCs
To confirm that preterm UC-MSCs are characterized by an
anaerobic metabolism, the preterm and term UC-MSCs, as
well as BM-MSCs have been grown in hypoxic condition
(1% oxygen tension for 48 h) (Fig. 8). Preterm UC-MSCs
grown in hypoxic condition showed comparable values of
ATP/AMP ratio and OXPHOS activity with respect the
same cells cultured in normoxia (panels a–c). Moreover,
also the TEM analysis did not show significant morpho-
logic changes (panel d). By contrast, term UC-MSCs (39
w) and BM-MSCs grown in hypoxia show a great impair-
ment of ATP/AMP ratio (panel a) and aerobic metabo-
lism (panels b, c). The low oxygen tension also caused
evident swelling of mitochondrial cristae, which appear
less-defined (panel d).
13
S. Ravera et al.
value of term UC-MSCs (39 weeks of GA) and BM-MSCs. c Reports
the expression of SOD2, an antioxidant mitochondrial enzyme. The
loaded samples are: preterm UC-MSCs (28, 32 and 34 weeks of GA),
term UC-MSCs (39 weeks of GA), and BM-MSCs used as positive
control. The densitometric analysis of the WB signals is shown in d.
c, d are representative of at least four experiments. Double asterisk
indicates a significant difference for p  <  0.001 among preterm UC-
MSCs with respect to the value of term UC-MSCs (39 weeks of GA)
and BM-MSCs
Metabolic changes in endothelial cells form umbilical
cord blood of preterm and term newborns
To evaluate whether other tissues display a metabolic shift
from anaerobic to aerobic metabolism, during the fetal
development, the energy metabolism was evaluated also in
endothelial cells.
WB analysis shows that preterm endothelial cells
(32 weeks of GA) displayed a lower expression of CLUH,
COXII, β subunit of ATP synthase, and OPA1, as reported
for preterm UC-MSC, with respect to the endothelial cells
of 36 and 38 weeks of GA (Fig. 9, panels a–e). The differ-
ence in the expression of OXPHOS proteins suggested that
preterm endothelial cells are characterized by an anaerobic
metabolism, which shifts to an aerobic respiration pro-
portionally to the GA. In particular, preterm endothelial
cells are characterized by a lower ATP/AMP ratio (Fig. 9,
Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic…
Fig. 5  Mitochondrial morphology and organization of mitochon-
drial reticulum in preterm and term UC-MSCs. a Reports the TEM
analysis to evaluate the morphology of mitochondria in preterm (28
and 35 weeks of GA), in term (39 weeks of GA), and BM-MSCs,
while table in b shows the number of mitochondria/cell, expressed
as mean  ±  SD. No relevant difference in mitochondrial morphol-
ogy or number is observable in the samples. c shows the structure
of mitochondrial reticulum, stained with mitotracker deep red. In
early preterm sample (28 weeks of GA), mitochondria appear punc-
tuated and not organized in a network. In preterm sample (34  w of
GA), the mitochondrial reticulum is more evident and becomes well
panel f), oxygen consumption (Fig. 9, panel g), and ATP
synthesis (Fig. 9, panel h) with respect to the term cells.
Moreover, also the lipid peroxidation (Fig. 9, panel k)
and catalase activity appeared diminished than in term
organized in term (39 weeks of GA) and BM-MSCs. d Reports the
expression of OPA1 and DRP1, involved, respectively, in the mito-
chondria fusion and fission. The loaded samples are: preterm UC-
MSCs (26–34 weeks of GA), term UC-MSCs (39 weeks of GA), and
BM-MSCs, used as positive control. The densitometric analysis of
the WB signals is shown in e. Each panel is representative of at least
four experiments. Double asterisk indicates a significant difference
for p < 0.001 among preterm UC-MSCs with respect to the value of
term UC-MSCs (39 weeks of GA), while asterisk indicates a signifi-
cant difference for p < 0.005 between term UC-MSCs and BM-MSCs
endothelial cells. This low OXPHOS activity is compen-
sated by a high glycolytic rate (Fig. 9, panel i) and lactate
production (Fig. 9, panel j).
13

Fig. 6  Expression of CLUH in preterm and term UC-MSCs. The fig-
ure shows the q-PCR (a) and WB analysis (b signals and c densito-
metric analysis) to evaluate the expression of CLUH in preterm (26,
28 and 34 weeks of GA) and term (39 weeks of GA) UC-MSCs. The
q-PCR data are representative of at least five experiments and asterisk
Discussion
Specific metabolic pathways involved in the energy produc-
tion influence cell functions. Two main pathways are active
in mammalian cells during development: the oxidative
phosphorylation (OXPHOS) and the anaerobic glycolysis,
which are finely regulated according to cell requirements
and specification.
OXPHOS that occurs in mitochondria represents the prin-
cipal site of aerobic energy production, synthesizing ATP by
oxygen consumption [30]. Anaerobic glycolysis is consid-
ered as a source of energy that could be advantageous either
when a low O ­ 2 consumption is required either in limited oxy-
gen availability conditions [31]. Even if anaerobic glycolysis
is less efficient than OXPHOS in terms of energy produced,
it does not determine an oxidative stress production, limiting
the structural damages [32] and the relative aging of the cell
[6]. The balance between anaerobic and aerobic metabolism
influences the chromatin structure, gene transcription, and
maintenance of pluripotency [33].
In this work, we have analyzed the energy metabolism of
preterm and term UC-MSCs, observing a metabolic switch
from anaerobic to aerobic metabolism, during fetal develop-
ment. In fact, oxygen consumption and ATP synthesis were
very low in early preterm samples and increased around the
34th week of GA at the expense of glycolytic flux (Fig. 3).
These data were particularly evident when the mitochondrial
function was stimulated with pyruvate plus malate, which
induces the OXPHOS activity through the complexes I, III,
and IV, the principal pathway of the electron transport chain.
In addition, the evaluation of OXPHOS efficiency showed
that in preterm UC-MSCs, oxygen consumption is not com-
pletely linked to ATP synthesis, indicating a partially uncou-
pling (Fig. 3). In fact, in a complete coupling condition,
the P/O value after the induction with pyruvate plus malate
should be around 2.5 [24], while in early preterm UC-MSCs,
the P/O value was around 1.6–2.1 (Fig. 3, panel c).This
less efficient mitochondria metabolism determines a high
13
S. Ravera et al.
indicates a significant difference for a p  <  0.005. The WB data are
representative of at least four experiments. Double asterisk indicates
a significant difference for p < 0.001 among preterm UC-MSCs with
respect to the value of term UC-MSCs (39 weeks of GA)
Fig. 7  Metabolic changes in term MSCs silenced for CLUH. a▸
Reports the WB analysis against CLUH, COX II, β subunit of ATP
synthase and OPA1 in term MSCs (CTRL), scramble MSCs (SCR)
and term silenced for CLUH (siRNA CLUH). b–e show the relative
densitometric analyses normalized on the actin signal showed in a.
The WB data are representative of at least three experiments. Double
asterisk indicates a significant difference for p < 0.001 among CLUH
silencing term UC-MSCs with respect to the value of term UC-MSCs
(CTRL). All the metabolic analyses were performed on term MSCs
(CTRL), scramble MSCs cells (SCR), and term MSCs silenced for
CLUH (siRNA CLUH). The energy status in terms of ATP/AMP
ratio is reported in f. The OXPHOS activity, as oxygen consumption
and ATP synthesis are shown in g and h, respectively. i Reports the
activity of hexokinase (HK), phosphofructokinase (PFK), pyruvate
kinase (PK), and lactate dehydrogenase (LDH), while j shows the
lactate released. The lipid peroxidation and the catalase activity are
reported in k and l, respectively. The organization of mitochondrial
reticulum was observed to confocal microscopy, using mitotracker as
fluorescence probe (m). Each panel is representative of at least three
experiment and double asterisk indicates a significant difference for
p < 0.001 among CLUH silencing term UC-MSCs with respect to the
value of term UC-MSCs (CTRL)
intracellular AMP concentration that decreases during the
GA progression (Fig. 2). However, both preterm and term
UC-MSCs contained a similar ATP intracellular concentra-
tion, indicating that, at any age, cells are able to produce
the necessary amount of energy to sustain their functions.
In fact, preterm UC-MSCs displayed a higher proliferative
potential in comparison with term UC-MSCs (Fig. 1), giving
rise to a large progeny when cultured in vitro. Our results
suggest that preterm MSC display a metabolic asset very
similar to what has been shown in ES or iPS that mainly use
a glycolytic metabolism without impairment of their prolif-
eration capacity [34].This pathway seems associated with
higher protection to oxidative stress and DNA damage [6].
The same results were observed by evaluating the energy
metabolism in endothelial cells isolated from umbilical cord
of preterm and term newborn (Fig. 9).
Interestingly, we observed that low aerobic metabo-
lism in preterm UC-MSCs is not due to an alteration of
Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic…

mitochondrial morphology or to a low number of mito- highly efficient, providing the necessary connection
chondria within the cell. Rather, it seems linked to the between biochemical parameters and qualitative features
lack of an organized mitochondria reticulum (Fig. 5), a fundamental for the functionality of living cells [35]. In
complex structure that renders the oxidative metabolism fact, only term UC-MSCs and BM-MSCs show such an

13

Fig. 8  Energy metabolism, oxidative status, and mitochondrial mor-
phology in preterm and term UC-MSCs grown in hypoxic condition.
All data show a comparison of preterm (28 weeks of GA) and term
(39  weeks of GA) UC-MSCs and BM-MSCs grown in normoxic or
hypoxic condition (1% ­O2, for 48  h). a, b report the mitochondrial
oxygen consumption and the ATP synthesis trough Fo–F1 ATP syn-
thase. To stimulate the pathway composed by complexes I, III, and
IV, pyruvate  plus  malate (Pyr/Mal; gray columns) was employed;
while succinate (white columns) was used to stimulate the pathway
organized mitochondria reticulum that is not observed in
preterm cells. The immaturity of the mitochondria appara-
tus was confirmed by the expression of OPA1 and DRP1,
two proteins involved in fusion/fission processes, which
increased proportionally with the GA (Fig. 5). Modifica-
tion of bioenergetic profile was observed also in MSCs
from mice, during the commitment, due to the changes
in mitochondrial morphology and fission/fusion process,
suggesting a central role for mitochondrial dynamics in
the maintenance/commitment of MSCs [36]. In particular,
mitochondrial elongation and an increased expression of
fusion proteins were observed in the early steps of adipo
and osteogenesis differentiation, while chondrogenesis has
been characterized by a fragmented mitochondrial phe-
notype, associated with an increment of fission proteins
expression [36].
These metabolic changes seem under the control of
CLUH, a cytosolic messenger ribonucleic acid (RNA;
mRNA)-binding protein involved in the mitochondria bio-
genesis and distribution inside the cell [20]. As shown in
Fig. 6, both the mRNA and the protein expression of CLUH
appeared less represented in preterm sample and increased
proportionally with the GA.
13
S. Ravera et al.
formed by complexes II, III, and IV. c shows the ATP/AMP ratio. d
Reports the TEM analysis. Mitochondria from UC-MSC (preterm)
patients appeared morphologically normal before and after hypoxia
treatments. On the contrary, both term than BM-MSC showed mito-
chondria with swelling cristae (white arrowheads) after hypoxia treat-
ments. (Scale bars, 1 μm). Each Panel is representative of at least five
experiments. Double asterisk indicates a p < 0.001 between the sam-
ple grown in hypoxia and grown in normoxia
To verify whether the metabolic switch during the fetal
development is led by CLUH, a silencing of this protein was
performed in term MSCs (Fig. 7). Data show that CLUH
siRNA-treated term MSCs displayed an energy metabolism
and a production of oxidative stress similar to that observed
in the preterm MSCs, confirming the hypothesis that the
hypo-function of mitochondria in preterm MSCs is linked
to the low CLUH expression. These results further extend
the observation by other authors [20, 29], who described an
association between CLUH expression and mitochondrial
function and corroborate the concept that CLUH favors the
organization of mitochondrial reticulum promoting the oxi-
dative metabolism.
The aerobic metabolism is always associated with an
increment of oxidative stress production [37, 38]. However,
the proportional increment of malondialdehyde (MDA)
level, during the metabolic switch, was balanced by the
catalase activity and the expression of SOD2 (Fig. 4), sug-
gesting that UC-MSCs have sufficient endogenous antioxi-
dant defenses. On the other hand, it is important to note that
early preterm UC-MSCs produce MDA, despite the high
glycolytic metabolism. This lipid peroxidation may be due
to the uncoupled OXPHOS metabolism, as demonstrated
Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic…
Fig. 9  Metabolic characterization of endothelial cells isolated from
umbilical cord of preterm and term newborns. a Reports the WB
analysis against CLUH, COX II, β subunit of ATP synthase, and
OPA1 in early preterm endothelial cells (32  weeks of GA), preterm
endothelial cells (36  weeks of GA) and in term endothelial cells
(38  weeks of GA). b–e show the relative densitometric analysis
normalized on the actin signal showed in a. The WB data are rep-
resentative of at least three experiments. Double asterisk indicates a
significant difference for p  <  0.001 among preterm endothelial cells
(32 weeks of GA) with respect to the value of term endothelial cells
by the low P/O value (Fig. 3). In fact, one of the causes of
the oxidative stress is the uncoupling between the electron
transport chain and the ATP synthase activity [39], because,
in this condition, the oxygen consumption rate considerably
increases, favoring the formation of reactive oxygen species
(ROS).
It is important to consider that the results described
herein have been obtained on UC-MSCs grown in nor-
moxic condition, while in vivo they reside in hypoxic
environments [40]. In fact, a comparison of arterial blood
pO 2 has shown that fetal oxygenation is close to the
(38  weeks of GA). All the metabolic analyses were performed on
early preterm endothelial cells (32 weeks of GA), preterm endothelial
cells (36 weeks of GA), and term endothelial cells (38 weeks of GA).
The energy status in terms of ATP/AMP ratio is reported in f. The
OXPHOS activity, as oxygen consumption and ATP synthesis are
shown in g, h, respectively. i Reports the activity of hexokinase (HK),
phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehy-
drogenase (LDH), while j shows the lactate released. The lipid per-
oxidation and the catalase activity are reported in k and l, respectively
values that determined a hypoxic condition in adult [41,
42]. This condition seems to play a pivotal role in the fetal
development [43, 44], considering that hypoxia plays a
critical role in the formation and development of the heart
[43], through the HIF modulation which regulates numer-
ous functions, such as energy metabolism, erythropoiesis,
cell survival and death, vascularization, angiogenesis, and
differentiation [44–46]. However, it was observed that
UC-MSCs expanded under normoxia seem to display a
high ­O2 consumption rate, suggesting that glycolysis may
be an environmental adaptation for UC-MSCs [47]. To
13

evaluate whether metabolic switch observed in UC-MSCs
depends by the growth conditions, experiments were per-
formed also in hypoxic condition (1% of ­O 2, for 48 h).
As reported in Fig. 8, the ATP/AMP ratio (panel c), the
OXPHOS activity (panels a, b), and the mitochondrial
morphology (panel d) of preterm sample did not show
significant difference in comparison with those main-
tained in normoxia. By contrast, in term and BM-MSCs
grown in hypoxic condition, the same parameters resulted
as severely impaired, suggesting that the metabolism of
these cells is “irreversibly oxygen-dependent”.
Therefore, if we consider the UC-MSCs as a model of fetal
cells [48, 49], data reported herein suggest that, in preterm
newborn tissues, the immature OXPHOS machinery, not able
to use correctly the oxygen, determines an enhancement of the
oxidative stress and increased inflammation response and tis-
sue damage. In fact, it is known that the exposure of preterm
newborns to oxygen leads to the accumulation of reactive oxy-
gen species (ROS), which results in the generation of hydro-
gen and lipid peroxides [50], causing tissue damages [51].
In conclusion, our study provides new information on the
production of energy and mitochondrial metabolism during
the passage from fetal to adult life, and discloses the changes
necessary to optimize the mitochondria function; moreover,
it extends the notion that CLUH plays a role in such organi-
zation. These data shed new light on the pathophysiology of
damages occurring in preterm newborns [51, 52] and may
provide useful information for the clinical management of
these patients to individuate new protective therapeutic strat-
egies aiming to reduce entity and number of complications
of prematurity.
Acknowledgements  This work was supported by funds from Cinque
per mille e Ricerca Corrente, Ministero della Salute, to Istituto Gian-
nina Gaslini; a Compagnia di San Paolo Grant (2014AAI637.U/812/
AR pv 2013.0958 to F.F) and a Grant FIRB (2012# RBFR1299K0_002
to C.F.). The authors are indebted to Dr. Federica Raggi for providing
the hypoxia incubator.
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