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Human placental mesenchymal stromal cells (pMSCs) have never been investigated in intrauterine growth restriction (IUGR). We characterized cells isolated from placental membranes and the basal disc of six IUGR and five physiological placentas. Cell viability and proliferation were assessed every 7 days during a 6-week culture. Expression of hematopoietic, stem, endothelial, and mesenchymal markers was evaluated by flow cytometry. We characterized the multipotency of pMSCs and the expression of genes involved in mitochondrial content and function. Cell viability was high in all samples, and proliferation rate was lower in IUGR compared with control cells. All samples presented a starting heterogeneous population, shifting during culture toward homogeneity for mesenchymal markers and occurring earlier in IUGR than in controls. In vitro multipotency of IUGR-derived pMSCs was restricted because their capacity for adipocyte differentiation was increased, whereas their ability to differentiate toward endothelial cell lineage was decreased. Mitochondrial content and function were higher in IUGR pMSCs than controls, possibly indicating a shift from anaerobic to aerobic metabolism, with the loss of the metabolic characteristics that are typical of undifferentiated multipotent cells.
This study demonstrates that the loss of endothelial differentiation potential and the increase of adipogenic ability are likely to play a significant role in the vicious cycle of abnormal placental development in intrauterine growth restriction (IUGR). This is the first observation of a potential role for placental mesenchymal stromal cells in intrauterine growth restriction, thus leading to new perspectives for the treatment of IUGR.
Mesenchymal cells are the predominant cellular component in placenta and serve as structural support for trophoblast villi and vascular network . In recent investigations, human placental mesenchymal stromal cells (pMSCs) have been isolated according to the meeting criteria proposed by the Mesenchymal and Tissue Stem Cell Committee  and the International Society for Cellular Therapy as cells that adhere to untreated plastic surfaces and express CD105, CD73, and CD90 but not CD34, CD14, CD19, CD11b, CD79α, or HLA-DR and that differentiate into osteogenic, adipogenic, and chondrogenic lineages in vitro [3, 4]. Both fetal (from placental membranes and villi) and maternal (from decidua) pMSCs can be isolated from the human placenta, showing similar phenotypes [5–8]. Indeed, the placental maternal side comprises the chorionic villi on one side and maternal decidua on the other. The peripheral region of the placenta on the maternal side that is in contact with the uterine wall (called the basal plate) comprises the chorionic villi on one side and maternal decidua basalis on the other. The significant issue of the presence of maternal cells in human pMSCs cultures was reviewed recently .
Despite progress in the characterization of pMSCs, it remains largely unknown to what degree the pMSCs are involved in placenta disease. Investigation of pMSCs may therefore give important hints for pregnancy pathologies linked to placental insufficiency, namely intrauterine growth restriction (IUGR) and preeclampsia (PE) . IUGR and PE are leading causes of maternal and neonatal morbidity and mortality [11, 12]. A specific placental phenotype has been associated with IUGR , characterized by altered transport of oxygen and nutrients [14–17] and impaired mitochondrial content and function [18, 19]. We recently showed altered mitochondrial content and functionality in cytotrophoblast cells isolated from IUGR placentas  suggesting compensatory mitochondrial mechanisms that occur under placental vascular insufficiency. Indeed, IUGR placentas present defective vascular transformation and terminal villous formation, with fewer and thinner villi and vessels, especially in the most severe cases, that are differently distributed along the placental surface, as reviewed by Gourvas et al. .
Only a few studies have focused on the role of pMSCs in placental insufficiency, and all of them have addressed preeclampsia [1, 21–25]. Human pMSCs isolated from PE placentas and deciduas display decreased proliferation and, interestingly, show proinflammatory and antiangiogenic characteristics [21, 24, 25] that lead to impairment of trophoblast development and invasive capacity and to defects in placental vasculogenesis. Currently, no data have been reported on pMSCs in IUGR.
Here we hypothesize that pMSC phenotype may be involved in the pathogenesis of IUGR. Thus, we performed a study to isolate human pMSCs residing in physiological and IUGR placentas and to characterize them in vitro, in reference to their immunophenotype, cell viability, expansion rate, differentiation potential, and mitochondrial content, in order to evaluate their potential involvement in IUGR placental phenotype.
Pregnant women were enrolled in the Unit of Obstetrics and Gynecology of the L. Sacco Hospital in Milan, Italy. The study was approved by the institutional ethics committee, and samples were collected with the informed consent of the patients. Human placentas were obtained at elective cesarean delivery (nonlaboring deliveries) from 11 singleton pregnancies.
We studied six pregnancies complicated by IUGR between 32 and 37 weeks of gestation. Gestational age was calculated from the last menstrual period and was confirmed by routine ultrasonography at 11–12 weeks of gestation. We identified growth-restricted fetuses in utero through repeated longitudinal measurements that demonstrated a reduction in fetal growth velocity [17, 26, 27]. Specifically, IUGR was defined by measurements of abdominal circumferences below the 10th percentile of reference values for fetuses of similar ages together with a shift from the growth curve greater than 40 centiles . As an example, this represents a decrease from the 50th centile (measured at 20 weeks) to below the 10th centile (at the time of IUGR diagnosis). Growth restriction was confirmed at birth if the neonatal weight was below the 10th percentile according to Italian standards for birth weight and gestational age .
Healthy controls (n = 5) were term (≥37 weeks) physiological pregnancies with normal intrauterine growth and appropriate for gestational age birth weight, according to reference ranges for the Italian population . Exclusion criteria were any placental or fetal disease. Indications for cesarean delivery before labor were breech presentation, previous caesarean deliveries, or maternal request. Exclusion criteria for both groups were maternal drug or alcohol abuse and autoimmune diseases. None of the fetuses had abnormal karyotype, genetic syndromes, viral infection, or major malformations.
Placental tissue was collected immediately after cesarean delivery and rapidly rinsed in phosphate-buffered saline (PBS) containing penicillin (200 U/ml) and streptomycin (200 μg/ml) for cell experiments. Placentas were weighed after discarding of the cord, membranes, and excess blood. Full-thickness pieces, 1.5 cm3, were sampled in different sites of the placental disc and washed in Hanks’ balanced saline solution (Sigma-Aldrich, St. Louis, MO, https://www.sigmaaldrich.com). After mechanical separation of placental membranes (PM) from the placental basal disc (PBD), these tissues were enzymatically digested with collagenase IA (Invitrogen, Life Technologies, ThermoFisher Scientific, Carlsbad, CA, https://www.thermofisher.com) and trypsin 2.5% (Invitrogen, Life Technologies, ThermoFisher Scientific, ) and incubated in a fully humidified atmosphere of 5% CO2, 95% air, at 37°C for 45 minutes.
Digested tissues were then filtered and centrifuged at 2000 rpm for 10 minutes, and cells were grown in expansion medium as previously described  with the following minor modifications. PM- and PBD-derived cells were plated in six-well tissue culture plates (VWR, Radnor, PA, https://vwr.com), coated with 0.2% gelatin (Sigma-Aldrich), at a density of 104 cells/well. Cells were grown in expansion medium composed of Dulbecco’s modified Eagle’s medium/F-12 (1:1) (Invitrogen, Life Technologies, ThermoFisher Scientific), 10% fetal bovine serum. and 20 ng/ml epidermal growth factor (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com/en). Expansion culture media were prepared fresh weekly. Cells were incubated in a fully humidified atmosphere of 5% CO2, 95% air, at 37°C for several weeks. Every week, living cells were counted with a Burker chamber using trypan blue (Sigma-Aldrich) exclusion method.
Cells were counted and passaged at a confluence of 70%–80% for up to 6 weeks of culture. At each passage, the population doubling (PD) rate was determined by using the formula doubling time = ln(2)/proliferation index, where the proliferation index is calculated as N(t) = the number of cells at time t/the number of cells at time of previous passages (t − 7days). The PD of each passage was calculated and added to the PD of the previous passages to generate the cumulative population doubling rate. All counts were performed in triplicate, and data are shown as mean fold (±SD in percentages of four experiments).
To assess proliferation and viability, samples were analyzed by MTT (3--2,5-diphenyltetrazolium bromide) activity assay  and expressed as percentage of viable cells on the total cell number. Values are expressed as means ± of three separate experiments.
PM- and PBD-derived pMSC cells (5 × 103) from physiological (n = 5) and IUGR (n = 6) placentas were used for the colony-forming unit-fibroblast (CFU-F) assay. Cells were plated in expansion medium in duplicate in six-well plates. After 14 days of culture in a humidified incubator at 37°C and 5% CO2, the colonies were stained with 1% crystal violet solution for 5–10 minutes and washed twice with deionized water. Aggregates of cells with a diameter between 1 and 8 mm were identified as colonies and counted under a light microscope (Leica DM6000R, Leica Microsystems, Renens VD, Switzerland, http://www.leica-microsystems.com/) using the software Image-J (National Institutes of Health, Bethesda, MD, http://imagej.nih.gov). pMSCs were also cultured in methylcellulose as described in the supplemental online data.
At 24 hours, 7 days, and 30 days, 105 cells were incubated with 7-amino-actinomycin D (7AAD; used to analyze only live cells [BD Biosciences/Pharmingen, San Jose, CA, https://www.bdbiosciences.com) and with the following antibodies: anti-CD34-allophycocyanin (APC) and anti-CD45-APC-CY7 (hematopoietic stem cell markers); anti-CD31-fluorescein isothiocyanate (FITC) (endothelial cell marker); and anti-CD44-FITC, anti-CD105-phycoerythrin (PE), anti-CD29-FITC, anti-CD73-APC, and anti-CD90-PE-Cy5 (mesenchymal cell markers) (Miltenyi Biotec). Isotype-matched mouse immunoglobulins were used as negative controls. After each incubation, performed at 4°C for 20 minutes, cells were washed in PBS, 1% heat-inactivated FCS, and 0.1% sodium azide. Cytometric analyses were performed with a Cytomics FC 500 flow cytometer and 2.1 CXP software (Beckman Coulter, Brea, CA, https://www.beckmancoulter.com). Each analysis included at least 10,000–20,000 events for each gate. A light-scatter gate was set up to eliminate cell debris from the analysis. The percentage of positive cells was assessed after correction for the percentage reactive to an equivalent isotype control.
PM- and PBD-derived pMSC cells (5 × 103) from physiological (n = 3) and IUGR (n = 2) placentas were tested by chromosome analysis and real-time polymerase chain reaction (PCR) for Y chromosome (SRY) as described in the supplemental online data.
After in vitro proliferation, human pMSCs were plated in 24-well tissue culture plates, coated with 10 μg/ml laminin (Sigma-Aldrich), in expansion medium. After 3–5 days (70%–80% confluence), they were switched in low-serum (DMEM, 2% horse serum) fusion-promoting medium for myogenic differentiation. After 14 days of culture, differentiated muscle cells were detected by immunostaining with antibodies against α-smooth muscle actin (α-SMA; 1:50 dilution, mouse anti-human; Sigma-Aldrich) and desmin (1:50 dilution, rabbit polyclonal; Abcam Inc., Cambridge, MA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. Images were captured using the Leica TCS SP2 confocal system (Leica Microsystems) at 18°C–20°C.
Osteogenic, chondrogenic, and adipogenic potential of pMSCs was verified through the use of specific differentiation kits: hMSC Osteogenic BulletKit, hMSC Chondro BulletKit, and hMSC Adipogenic BulletKit (Lonza, Basel, Switzerland, http://www.lonza.com) (supplemental online data).
Expanded human pMSCs were incubated in an endothelial growth medium containing medium 199 (Gibco-BRL, Life Technologies, ThermoFisher Scientific) supplemented with 50 ng/ml vascular endothelial growth factor (VEGF). Cells were seeded at a density of 30,000 cell/cm2 on six-well plates coated with Matrigel (Corning, Corning, NY, https://www.corning.com/medium)/199 medium (1:1) and incubated in a humidified atmosphere with 5% CO2, 95% air, at 37°C. After 24 hours, we observed the formation of capillary-like tube structures by using an inverted microscope (Leica DMIRE2). Manual tube identification was performed using ImageJ software. To depict the beginning and ending points of individual tubes on the processed image, three different operators processed four selected images. As result of this process, a tube topology graph was formed that enables the gathering of valuable statistics, such as total tube length. Tube length was compared in normal and IUGR differentiated pMSCs. Human fibroblast cells were used as a negative control. To confirm tubulogenesis, immunostaining with antibody against von Willebrand factor (vWF; diluted 1:50; Dako, Glostrup, Denmark, http://www.dako.com) was performed on capillary-like tube structures. Images were captured by using an inverted microscope (Leica DM 6000R). Endothelial differentiation was confirmed by real-time PCR analysis of CD31 expression levels. Each experiment was performed in triplicate, and for each sample, the median value between the three experiments was reported.
We investigated the angiogenic potential of pMSCs isolated from physiological and IUGR PM and PBD by using a directed in vivo angiogenesis angioreactor (DIVAA) as described in supplemental online data .
pMSCs were analyzed for the expression of mitochondria-related genes after approximately 30 days of culture in expansion medium. Total RNA was extracted by TRIzol Reagent (Ambion, Life Technologies, ThermoFisher Scientific). After DNase I treatment (New England Biolabs, Ipswich, MA, https://www.neb.com), aimed at removing potentially contaminating DNA, 1.6 μg of total RNA were reverse-transcribed by using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies, ThermoFisher Scientific) with random examers. Gene expression of UQCRC1, COX4I1 (two subunits respectively belonging to the III and IV respiratory chain complexes), and NRF1 (a mitochondrial biogenesis activator) was analyzed by real-time PCR using the 7500 Fast Real-Time PCR System with TaqMan assays (Applied Biosystems, Life Technologies, ThermoFisher Scientific). Expression levels were determined according to the E-ΔCt/NF formula (GeNorm method ), which considers the assay amplification efficiency (E, calculated from a serial dilution curve) and a normalization factor (NF) relative to PPIA and RPL13A control genes. All samples were reverse-transcribed in duplicate, and cDNA was run in triplicate.
Individual clinical data of patients included in this study are presented in supplemental online Table 1. No significant differences emerged between groups for maternal age and prepregnancy BMI. As expected, gestational age, and placental and fetal weights were significantly lower in IUGR than physiological pregnancies.
Cells were isolated from PM and PBD tissues and cultured in expansion medium. PM- and PBD-derived cells adhered to plastic and showed a typical fibroblastic morphology of MSC by light microscopy (Fig. 1A–2D).
Given the risk for cross-contamination between fetal and maternal cells, we first analyzed physiological (n = 3) and IUGR (n = 3) PM- and PBD-derived cells by fluorescence in situ hybridization (FISH) to verify that pure cell populations had been isolated. For these experiments, PM- and PBD-derived cells were isolated from placentae of women delivering a male baby, and approximately 200 cells of each type were analyzed for signals in interphase nuclei using X/Y chromosome probes. Cells derived from PM showed the XY phenotype (spectrum green and orange-labeled chromosomes, respectively) and were therefore 100% male (Fig. 1E, ,1F)1F) as previously described . Two spectrum green-labeled X chromosomes were visible in cells derived from PBD with a low contamination (<5%) of XY phenotype; therefore, these cells were prevalently of maternal origin (Fig. 1G, ,1H),1H), in agreement with the literature [9, 35]. Real-time PCR with primers for the SRY gene confirmed these data (data not shown).
High cell viability was reported for 6 weeks (P5) for all the analyzed samples, confirming appropriate culture conditions (Fig. 1I). Cell proliferation was also monitored by using the MTT assay. After three passages (P3), PM- and PBD-derived cells isolated from physiological and IUGR placentas showed similar rate of proliferation (Fig. 1J, ,1K)1K) and became more homogeneous following expansion with a fibroblastic morphology characteristic of MSCs (Fig. 1). Otherwise, in long-term culture conditions (for 35 days; after P5) the IUGR PM- and PBD-derived cells displayed a lower proliferation rate and doubling time compared with healthy controls (Fig. 1L). Because placenta cultured cells were morphologically indistinguishable after passaging from MSC phenotype, it was crucial to characterize these cells for the MSC surface marker expression. Flow cytometry analysis demonstrated that 24 hours after isolation, all samples presented a heterogeneous population composed of different cell types (Figs. 2, ,3),3), whereas after 7 days of culture PM and PBD cells acquire a homogenous immunophenotype. In particular, the number of contaminating hematopoietic cells expressing the CD34 and CD45 markers progressively diminished to less than 1% after passaging (Fig. 2), with more cells positive for CD105, CD29, CD73, and CD44 mesenchymal markers (Fig. 3), indicating a progressive enrichment of pMSC.
To verify the presence of MSCs, PM- and PBD- (n = 3 per group) derived cells from IUGR and physiological placentas at P3 were assayed for their capacity to develop colony forming unit fibroblast (CFU-F) colonies (Fig. 1M–1O). Few colonies were counted in all samples with a non-statistically significant prevalence in physiological versus IUGR PM CFU-F (23.83 ± 6.24 vs. 17.58 ± 5.55; p = .07), whereas a statistically significantly increased number of colonies was found in physiological versus IUGR PBD CFU-F (37.89 ± 4.1 vs. 8.36 ± 5.75; p < .0001). Methylcellulose colony assays demonstrated the absence of hematopoietic colony forming unit granulocyte (CFU-G), colony forming unit-monocyte (CFU-M), and colony forming unit-granulocyte macrophage (CFU-GM) colonies after P3 (data not shown), suggesting a simultaneous loss of hematopoietic stem cell and pMSC enrichment in our culture system.
The enrichment of pMSCs occurred earlier in IUGR than in healthy samples (Fig. 3A–3F): (a) PM-derived cells from IUGR and healthy samples were CD105/CD29 (18.8% ± 1% and 10% ± 2%, respectively; p < .05) and CD73/CD44 (50% ± 47% and 5% ± 1% respectively, p < .05), and (b) PBD-derived cells from IUGR and healthy samples were CD105/CD29 (9.5% ± 1% and 1.6% ± 0.2% respectively; p < .05) and CD73/CD44 (4% ± 0.5% and 2% ± 0.1% respectively; p < .05) (Fig. 3). After 7 days of culture, the percentage of PM- and PBD-derived cells with mesenchymal immunophenotype was, respectively, 1.6–4-fold (Fig. 3C) and 2.3–5.8-fold (Fig. 3F) higher in IUGR compared with healthy controls. Differences turned out to be statistically significant for CD105, CD44, CD73, and CD90 (p < .05) and for CD29 (p < .001) expression in PM-derived cells (Fig. 3C) and only for CD29 (p < .05) in PBD-derived cells (Fig. 3F). After 30 days of culture, all PM- and PBD-derived cells were positive for typical MSC markers CD44, CD105, CD29, CD90, and CD73 (Fig. 3A–3F) and were negative for CD31 endothelial marker (data not shown). Overall, the data were consistent with the expression profile stipulated for pMSCs.
Differentiation of expanded pMSCs from PD and PBD of healthy and IUGR placentas into osteogenic, adipogenic, and chondrogenic lineages was examined to further verify their in vitro MSC properties. Alizarin red-stained calcium deposits indicative of bone formation were visible in pMSCs of all samples maintained in osteogenic induction medium (Fig. 4A). Oil Red O-stained lipid droplets were observed around cell nuclei in pMSCs stimulated in adipogenic differentiation medium (Fig. 4A–4C). In this condition, the IUGR pMSCs differentiated 1.7-fold more than did physiological ones, as confirmed by the number of cells expressing fatty acid binding protein 4 (FABP4) and perilipin A (data not shown). In chondrogenic conditions, the pMSCs were positive for alcian blue staining (Fig. 4A); however, no significant differences were observed between the healthy and IUGR PD and PBD groups. To determine whether IUGR affected pMSC multipotency more broadly, we evaluated the ability of pMSCs to differentiate into other cell types, specifically muscle cells. Human desmin and α-SMA-positive myotubes were formed in both IUGR and physiological groups on day 10. No significant differences were observed in the percentages of desmin-expressing cells/total nuclei when the two groups were compared (data not shown). Collectively, these in vitro findings demonstrate the multipotency of healthy and IUGR-derived pMSCs and a prevalence of adipocyte differentiation in IUGR pMSCs.
Under endothelial growth medium, we observed thicker capillaries, creating a complex network predominantly in PBD-derived pMSCs (Fig. 5A). The specificity of the observed capillary structures was confirmed by their complete positivity for vWF and negativity of fibroblast cells used as negative control (Fig. 5A). Moreover, endothelial tubular structures were highly reduced in PM- and PBD-derived pMSCs from IUGR compared with healthy specimens (Fig. 5A). Indeed, the formation rate of the tubular structures during 72 hours of culture and their length (Fig. 5B) remained significantly lower in IUGR pMSCs than those in physiological ones (p < .05). Real-time PCR analysis confirmed statistically significantly higher CD31 expression (p < .05) in physiological compared with IUGR differentiated pMSCs (Fig. 5C).
The angiogenic effect of pMSC isolated from physiological and IUGR placental membranes and placental basal discs was evaluated indirectly by incubating pMSC-conditioned media with a human umbilical vein endothelial cell (HUVEC) cell line. After 24 hours, we observed a higher, non-statistically significant formation of capillary-like tube structures in PBD than PM-derived medium conditions (Fig. 5D). To further extend these in vitro data, we performed in vivo experiments using a previously described reproducible and quantitative angiogenesis assay (DIVAA system) . Fifteen days after subcutaneous implantation in nude mice, the angioreactors containing IUGR and physiological pMSCs showed numerous blood vessels positive for vWF expression (Fig. 6). A well-organized network of vessels was found in the fibroblast growth factor-2-positive control (8.8 ± 2.4) and in the samples containing pMSCs derived from PM and PBD physiological (27.4 ± 8.4 and 25.3 ± 3.2 respectively) and IUGR (10.6 ± 2.8 and 8 ± 1.6 respectively) samples (Fig. 6). Few endothelial structures were also found in the Matrigel-negative control group (5 ± 1.4) (Fig. 6). All these data suggest an impaired endothelial potential of IUGR pMSCs.
Because we observed a reduced endothelial potential in IUGR pMSCs, we hypothesized that environmental placental hypoxia may influence the respiratory mitochondria gene expression of these cells. Indeed, recent literature demonstrated that mitochondrial dysfunction also represents a critical factor for fetal programming in cases of placental insufficiency . We therefore investigated the expression levels of the respiratory chain genes UQCRC1 and COX4I1 and of the mitochondrial biogenesis activator NRF1 in IUGR (n = 3) and physiological (n = 3) pMSCs. IUGR compared with physiological pMSCs displayed a trend toward higher levels of all analyzed genes in the PBD and of COX4I1 in PM groups (p < .05) (Fig. 7). These data may suggest a role of the pMSCs in IUGR and are in agreement with our previously reported results .
Here we report for the first time pMSC proliferation and differentiation characteristics in human IUGR pregnancies. Mesenchymal marker enrichment and multipotent differentiation abilities confirmed successful isolation and selection of a mesenchymal stromal cell population from membranes and basal disc of both physiological and IUGR placentas. As attested by flow cytometry data, the pMSC population was selected earlier in IUGR placentas, although initial cell type proportions were similar in the two groups. Moreover, this population displayed lower endothelial and higher adipogenic differentiation potentials compared with normal controls. We found lower proliferation rate in IUGR pMSCs compared with controls, especially after 35 days of culture.
A significant decrease in pMSC proliferation rate has been previously reported in PE with fetal and/or placental compromise compared with physiological pregnancies . Authors hypothesized that this pMSC reduction could impair primary villi formation and trophoblast development during placentation because pMSCs both serve as structural support and exert a paracrine activity on trophoblast cells. Thus, this could contribute to the abnormal villous architecture found in PE [1, 24]. Decreased proliferation was also found in MSCs isolated from PE deciduas, and linked to microRNA-16 (miR-16) overexpression leading to cyclin E1 downregulation. High miR-16 levels also caused VEGF subtype A and decreased secretion and led to consequent reduction of trophoblast migration, HUVEC migration, and blood vessel formation. It was therefore suggested that abnormal decidual MSCs overexpressing miR-16 may be involved in PE development by altering the pregnancy microenvironment that influences angiogenic processes .
As attested by flow cytometry, in IUGR placentas the mesenchymal stromal cell population is selected earlier than seen with controls. This suggests that in the pathological tissue-specific conditions might occur so that mesenchymal stromal cells are more resistant and selected faster. However, IUGR pMSCs showed a low rate of proliferation and altered differentiation capacities toward endothelial and adipogenic lineages. Thus, the faster selection of the mesenchymal cell type in IUGR cultures might represent a compensatory mechanism to metabolic alterations occurring in IUGR placental cells and/or to the adverse IUGR placental environment.
Our data showed lower endothelial differentiation potential of pMSCs isolated from IUGR placentas compared with healthy controls. Placental mesenchymal cells have been proposed to play an important role in placental vascularization during the whole human pregnancy by contributing both to vasculogenesis and angiogenesis, which are critical for placental development . Abnormal vasculogenesis and angiogenesis are correlated with the impaired placental and fetal development observed in complicated pregnancies, such as IUGR . Interestingly, in vitro studies showed that IUGR placental villous stromal cells altered ability to stimulate endothelial tubule-like structure formation and migration . In addition, the imbalance between pro- and antiangiogenic factors is well documented in placentas and in maternal blood of IUGR and PE pregnancies, suggesting a role in their pathophysiology [38, 39]. Endothelial progenitors contribute to tissue regeneration by induction of new vessels, thereby increasing tissue perfusion and oxygenation [40, 41]. The formation of these networks is important for organogenesis during fetal development. Indeed, endothelial progenitor cells play a critical role in blood vessel formation and vascular homeostasis and can also lead to tissue disorganization and fibrosis, as reviewed by Piera-Velazquez et al. .
Interestingly, several studies have pinpointed alterations in maternal and fetal endothelial progenitor cells [43, 44]. The mechanisms underlying impaired neovascularization in feto-placental circulation occurring in IUGR still need to be clarified and may be associated with the reduced capacity of pMSCs to give rise to differentiated endothelial cells. Unlike endothelial differentiation capacity, we found increased adipogenic potential in IUGR pMSCs compared with controls. To our knowledge, pMSC adipogenic potential has never been quantitatively assessed in the pathologic human placenta. However, IUGR newborns have increased risk for adult obesity and metabolic syndrome .
In animal models of IUGR, several alterations involving both the adipose tissue structure and endocrine system have been demonstrated. Changes in central nervous system appetite control and hormone secretion and regulation, as well as an increase of adipocyte differentiation, have been shown in IUGR rat offspring, as reviewed by Desai and Ross . Because these changes are evident early in life, the predisposition to obesity may be programmed in utero, as already hypothesized in the “fetal programming” theory , possibly through altered epigenetic mechanisms as recently reported in rat offspring . Mitochondrial dysfunctions also represent a critical factor for fetal programming in cases of placental insufficiency . Diaz and colleagues showed the link between fetal growth restraint and placental mitochondrial dysfunction, as reflected by changes in mitochondrial DNA (mtDNA) content and superoxide dismutase activity .
Because we previously demonstrated that mtDNA content was high in IUGR placentas , we investigated the gene expression levels of the mitochondrial respiratory chain genes UQCRC1 and COX4I1 (respectively belonging to the III and IV respiratory chain complex) and of the mitochondrial biogenesis activator NRF1 of IUGR and healthy pMSCs. Mesenchymal stem cell metabolism is known to be mainly anaerobic. The shift toward an aerobic mitochondrial metabolism is reported during differentiation, with increase of mitochondrial biogenesis, oxygen consumption, and ATP production . Thus, mitochondrial modifications might account for the higher adipogenic and the lower endothelial differentiation ability of pMSCs derived from IUGR placentas. Interestingly, pMSCs cultured with no differentiating medium presented a trend toward higher NRF1, UQCRC1, and COX4I1 expression levels in IUGR basal disc samples compared with controls and higher COX4I1 levels in IUGR placental membranes. These differences were not statistically significant, probably because of the low sample number. Nevertheless, they might account for metabolic alterations in IUGR pMSCs, showing a possible shift to aerobic metabolism, with the loss of the metabolic characteristics that are typical of multipotent and undifferentiated cells. This might be due to differences in oxygen concentrations within the placenta, occurring in IUGR [50, 51], regulating mitochondrial biogenesis and function, as previously observed in other animal tissues [52, 53].
We recently showed higher mitochondrial content in IUGR placental tissue; this content was decreased in cytotrophoblast cells isolated from the same placentas [18, 19]. We hypothesized that other cell types may be responsible for the mitochondrial content increase in the entire placental tissue, despite the decrease in cytotrophoblast cells. Here we suggest that pMSCs might contribute to the global mitochondrial content increase in IUGR compared with control placental tissues, possibly together with other placental cells, such as endothelial cells.
A potential limitation of this study is represented by the different gestational age of the IUGR compared with the control group. This is a limit of all studies comparing human IUGR to control term placentas because IUGR pregnancies are electively delivered earlier in the interest of the fetus. Another potential limitation is that we investigated IUGR placentas at delivery, whereas placental abnormal development is supposed to start at the beginning of placentation. Therefore, a cause-effect relationship may not be inferred because the observed features might also be the consequence of the altered placental environment influencing pMSCs. In the developing embryo, subtle changes in the microenvironment drive epigenetic change associated with cellular differentiation . However, this inherent sensitivity to external influence, which is a hallmark of early development, potentially renders epigenetic marks of pMSCs susceptible to external environmental influence. At present, no strong evidence exists for a role of epigenetic disruption as a driver of IUGR .
Interestingly, we demonstrated a fetal origin of the PM-derived pMSCs and a maternal low contamination of PBD pMSCs. These data agree with the literature [34, 56] and pointed out whether maternal pMSCs may be involved in IUGR disease. Thus, further studies are required to better understand the role of pMSCs in IUGR and how environmental influence on placental functioning modulate their regenerative capacities.
The observed IUGR-specific features may indicate a role of pMSCs in determining the specific placental phenotype associated with IUGR pathogenesis. The loss of endothelial differentiation potential and the increase of adipogenic ability that we report are likely to contribute to the vicious cycle of abnormal placental development in IUGR. These genuine new findings fit well with what is already known about placental pathological features in IUGR. Targeting pMSCs may thus lead to new perspectives for the treatment of IUGR.
We thank all the midwives and nurses of the Unit of Obstetrics and Gynecology of the L. Sacco Hospital in Milan for their competence and cooperation. We also thank all the pregnant patients for participating in the study. This work was financially supported by grants from Fondazione Giorgio Pardi and from the Italian Ministry of University and Research PRIN 2010-2011 prot. 20102chst5_005 “Parto pre-termine: markers molecolari, biochimici e biofisici dell’unità feto-placentare” (to I.C.). It was also supported by Associazione Amici del Centro Dino Ferrari, Associazione Amici di Emanuele, and Fondazione Opsis Onlus (to Y.T.).
C.M.: conception and design, provision of study materials or patients, manuscript writing; P.R.: collection and/or assembly of data, data analysis and interpretation; C.N.: collection and/or assembly of data; G.M.A., S.E., S.B., M.M., A.T., M. Baccarin, A.R., and S.M.: performance of experiments; M. Belicchi: data analysis and interpretation, manuscript writing; Y.T.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript; I.C.: conception and design, provision of study materials or patients, data analysis and interpretation, manuscript writing, final approval of manuscript.
The authors indicated no potential conflicts of interest.