Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy and can be isolated from various sources of human adult tissues such as bone marrow (BM-MSC) and adipose tissue. However, cells from these tissues must be obtained through invasive procedures. We, therefore, characterized MSCs isolated from fresh placenta (Pl-MSC) and fetal membrane (Mb-MSC) through morphological and fluorescent-activated cell sorting (FACS). MSC frequency is higher in membrane than placenta (2.14% ± 0.65 versus 15.67% ± 0.29%). Pl/Mb-MSCs
Multipotent mesenchymal stem cells (MSCs) are able to self-renewed and differentiate into mesodermal lineages such as adipogenic, chondrogenic, osteogenic, myogenic, and angiogenic cells [
The bone marrow is the traditional source of human MSCs, but they have been isolated from a wide variety of human adult tissues such as adipose tissue [
Osteoactivin (OA) has the ability to regulate cell proliferation, adhesion, differentiation, and synthesis of extracellular matrix proteins in various cell types [
In this study, we optimized the isolation of placental and amniotic membrane MSC and compared their proliferative and differentiation potential to BM-MSCs. We isolated through different methods MSCs from placenta and fetal membranes, and we qualified them according to the standardize protocols from the international society for cellular therapy (ISCT) [
Following approval from the Internal Review Board (HMC-IRB Protocol 9109/09, Weill Cornell Medical College in Qatar), placentas and fetal membranes were collected from donors at Woman’s Hospital at Hamad Medical Corporation immediately after elective caesarean section at term in the absence of labor, preterm rupture of membrane, chorioamnionitis, preeclampsia, intrauterine growth retardation, or chromosomal abnormalities. The specimen were completely deidentified and considered as biological waste. Therefore, no consent form was taken from the patients.
Supplementary Figure 1 (available at doi:10.1155/2012/658356) depicts the isolation procedures used in this study. For placenta, the decidua basalis was removed prior to harvesting the placental tissues. The placenta parts were free of any fetal membrane. For fetal membrane, we decided not to separate the amnion and chorion parts to illustrate the most direct workflow for MSCs isolation. The harvested pieces of tissues were washed in phosphate buffer saline (PBS, PH 7.4), mechanically minced into pieces of approximately 1 mm2, and subsequently digested with dispase (1 mg/mL, Hyclone), collagenase (300 U/mL) (Hyclone), hyalluronidase (100 U/mL, Hyclone), and DNAse I (80 U/mL, Roche) for 1 h at 37°C under agitation (150 rpm). The homogenate was subsequently washed in PBS. Cells were then filtered on a 100
Two million viable cells were then either directly plated in MSCs culture media (DMEM low glucose with 20% FBS, 2 mM L-Glutamin, and 1% penicillin/streptomycin [
Several bone marrows MSCs from different donors (BM-MSCs) were purchased from Stem Cell Inc. (number MSC-001F, Stem Cell Inc.) and PromoCell (number 12974, PromoCell) and maintained in the same culture conditions as placenta-/membrane-derived MSCs (Pl/Mb-MSCs). We performed all analysis at the 4th passages in order to obtain a homogenous cell population and sufficient number of cells to perform all analysis in parallel.
For flow analysis of cell surface antigens and cell sorting, MSCs were stained for the expression of CD45, CD34, CD73, CD105, CD90, and CD29 using Mouse anti-human CD45 antibody (BD Biosciences, number 339192, clone 2D1) coupled with Amcyan, Mouse anti-human CD34 (BD Biosciences, number 555821, clone 581) coupled with FITC, Mouse anti-human CD105 (biolegend, number 323212, clone 43A3) coupled with AF647, Mouse anti-human CD73 (BD Biosciences, number 550257, clone AD2) coupled with PE, Mouse anti-human CD29 (biolegend, number 323212, clone TS2/16) coupled with APC-Cy7, and Mouse anti-human CD90 (BD Biosciences, number 550402, clone 5E10) coupled with AF700.
Briefly, 1.106 cells were harvested and nonspecific sites were blocked in PBS-5%FBS-1%BSA-10%FcR Blocking Reagent (Myltenyi Biotec) for 30 minutes on ice. Cell suspension was incubated with specific antibodies for 45 minutes on ice. After washes in PBS and filtration on 45
Cells in culture were grown on 8 chamber slides (BD falcon, number 354102) and stained as follows.
The antibodies used were Mouse anti-human CD29-FITC (biolegend, number 303016, clone TS2/16), CD73-PE (BD Biosciences, number 550257, clone AD2), CD90-AF568 (BD Biosciences, number 550402, clone 5E10), CD34-PE (BD Biosciences, number 555822, clone 581), CD45-Amcyan (BD Biosciences, number 339192, clone 2D1), unconjugated, and CD105 (BD Biosciences, number 555690, clone 266) revealed by a secondary goat anti mouse IgG1 antibody (invitrogen, number A-21121).
Briefly, nonspecific sites and Fc receptors were blocked with PBS/0.3% bovine serum albumin/0.5% HS for 30 minutes and FcR blocking reagent (Miltenyi, number 120-000-442). Sections were incubated with primary antibodies (1 hour 30 minutes), washed twice in PBS/0.5% Tween 20 (Sigma-Aldrich), and if necessary incubated with secondary antibodies (1 hour, AF488 goat anti-mouse IgG1 at 0.5
Adipogenic differentiation was induced by culturing 80% confluent MSC for 3 weeks in DMEM-HG, 1
Osteogenic differentiation was induced by culturing 90% confluent MSC for 3 weeks in DMEM-LG, 10% FCS, 0.1
Three days after MSCs plating (50% confluence), recombinant human osteoactivin was delivered in a single dose of 100 ng/mL in differentiation media DMEM-LG, 10% FCS, 0.1
RNA was isolated using Trizol reagent followed by additional purification using RNAeasy extraction kit from Qiagen (QIAGEN, number 74106) with RNA yields that produces satisfactory microarray data. Two quality control measures were carried out: (1) a spectrophotometric analysis and (2) a size fractionation procedure using a microfluidics instrument (Agilent Technologies). 200 ng of total RNA were analyzed on Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Data were analyzed using Parteck Software (V6.09.1110-6; Affimetrix). Class comparison between BM-MSCs and Pl/Mb-MSCs (three biological replicates of each) was performed to identify gene expression changes with a significant expression differences (
We used Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA) to identify and analyze relevant pathways from the gene lists obtained after comparison of BM-MSC and Mb/Pl-MSC. Networks were constructed by overlaying the genes in the gene list onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge database using keywords such as organ formation and osteoblast differentiation. Networks of the genes up- or downregulated Pl/Mb-MSCs as compared to BM-MSCs were then algorithmically generated based on their connectivity. A network is a graphical representation of the molecular relationships between genes. Genes are represented as nodes, and the biological relationship between two nodes is represented as a line. All edges are supported by at least one reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways knowledge database.
MSCs were cultivated in differentiation or control media in presence or absence of OA for 4 h. Cells were harvested and proteins extracted as recommended and quantified based on sample absorbance at 280 nm using nanodrop device (Thermo-Scientific). 200
Arrays were revealed using HorseRadish Peroxidase (HRP) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Data were collected using Geliance CCD camera (Perkin Elmer) and extracted using Image J software (NIH). Briefly, arrays’ pictures were inverted and background subtracted. We defined a 120-micron diameter area for signal capture. Median pixel density was used to evaluate the signal. For comparison, independent array values were normalized on their positive control intensity values.
MSCs were cultivated in serum free media for 72 hours as previously published [
Student-
Supplementary Figure 1 depicts the workflow chart of the enzyme-mediated cell isolation of human term placenta/membrane, by direct culture or cell sorting, for derivation of fibroblast-like cells, that we characterized as multipotent mesenchymal stem cells (MSCs). Two methods were used to isolate MSCs. (i) selection in specific MSC media after direct culture of cell suspension obtained following tissue digestion, (ii) fluorescent-activated cell sorting according to expression of specific MSCs markers as defined by the international society for cellular therapy (ISCT, positive for CD105, CD73, CD90, CD29, and negative for CD45 and CD34) [
We quantified the number of MSCs in fetal membrane and placenta using polyvariate flow cytometry. CD45−, CD34− cells were selected and analyzed for the expression of CD105, CD73, CD90, and CD29. MSCs as defined by these 4 markers were significantly more abundant in the membrane, Mb-MSC 15.67% (±0.29%) than the placenta, Pl-MSC 2.14% (±0.65%, Figure
Higher proportion of MSCs can be isolated from the membrane compare to the placenta. Fetal tissue derived MSCs display greater proliferation kinetic than bone marrow derived MSCs. (a) MSCs were defined as CD45−, CD34−, CD29+, CD90+, CD73+, and CD105+. Their proportion was then calculated in freshly digested placenta specimens and fetal membranes form 3 different donors. MSCs represented 15.67% (±0.29%) and 2.14% (±0.65%) of cells isolated from the membrane and placenta, respectively (**
The same results were found when 2 million cells were directly plated after tissue digestion. The number of adherent cells was significantly higher at day 1 in the membrane compare to the placenta (Figure
Growth kinetic of Mb-MSCs and Pl-MSCs were compared with BM-MSCs at the same passage. The proliferation rate of Pl/Mb MSCs was significantly higher than BM-MSCs (Figure
We first defined our cell population as being negative for CD45 and CD34. In all our independent experiences, the vast majority if not all (85% to 99%) of CD73+, CD105+ cells was also expressing CD90 and CD29 reaching the canonical definition of MSCs [
The Cell analysis strategy allows the isolation of 4 different cell populations. Cells were stained with mouse anti human CD45-Amcyan, CD34-FITC, CD29-APC-CY7, CD90-AF700, CD73-PE, CD105-AF647. After FSC-A/SSC-A selection, only CD45− and CD34− cells were considered. CD73, CD105, CD90, and CD29 profiles were then analyzed. CD73+ CD105+ cells were more than 85% to 98% positive for CD90 and CD29 (lower panel). However, CD90+ CD29+ represented a more heterogenous population when looking at CD73, CD105 stainings. We defined different cell populations based on CD90 and CD29 subpopulation: CD90+ CD29+; CD90− CD29+; CD90+ CD29−; CD90− CD29− (middle panel).
None of the CD90− CD29− cells were able to grow in MSCs media. The CD29− CD90− is actually a very homogenous population containing mostly CD73− CD105− cells (Supplementary Figure 2). In contrast, we were able to derive mesenchymal like cells from the 3 other sorted subpopulations. This indicates that expression of at least one of these 2 markers is indispensable for MSCs isolation and qualification.
After 4 passages, the large majority of the cells sorted expressed CD90 and CD29. (Table
Proportion of cell expressing MSCs specific markers. After 4 passages, every cell sorted subtype and bone marrow MSCs were stained for CD45, CD34, CD90, CD29, CD105, and CD73. 99% of the cells were CD34 and CD45 negative and more than 80% positive for the MSCs makers.
CD90+ | CD29+ | CD105+ | CD73+ | |
---|---|---|---|---|
Bone marrow | 97.6% | 90.9% | 99.7% | 98.7% |
Membrane | 87.6% | 99.8% | 96.7% | 99.8% |
Membrane CD29+ CD90+ | 79.3% | 99.7% | 96.4% | 99.5% |
Membrane CD29+ CD90− | 91.8% | 98% | 98.1% | 97.8% |
Membrane CD29− CD90+ | 85.2% | 99.9% | 98.5% | 99.4% |
Placenta | 91.1% | 99.8% | 99.2% | 99.5% |
Placenta CD29+ CD90+ | 79.1% | 99.9% | 99.1% | 99.9% |
Placenta CD29− CD90+ | 84.7% | 99.6% | 99.8% | 99.5% |
Specific induction of adipogenic and osteogenic differentiation was performed on Mb/Pl MSCs sorted based on CD29 and CD90 expression or directly plated after isolation and compared to BM-MSCs differentiation (Figure
Differentiation assay of Pl/Mb-MSCs in comparison to BM-MSCs and cytokines expression. (a) Representative differentiation of Pl/Mb-MSCs passage 4 is shown. Cells were kept in induction medium (differentiation) or control standard medium (control). (a–d) Osteogenic and adipocyte differentiation and control for BM-MSCs. (f–h) Osteogenic and adipocyte differentiation and control for Mb-MSCs. (b) Cytokine expressions of Mb-MSCs and BM-MSCs using the proteome profiler. (c) Quantification of cytokine optical density. Measurements were obtained with image J software (NIH).
All different cell populations from placenta or membrane regardless of the isolation protocol were identically able to differentiate into adipocytes and osteoblasts confirming their phenotypic and functional similarity (data not shown).
Cytokine secretion profile was highly similar between fetal and BM-MSCs with strong secretion of GRO
We first analyzed differences between membrane-derived and placenta-derived MSCs. As demonstrated by our PCA analysis, MSCs derived from membrane or placenta could not be differentiated based upon their transcriptomic profile (Figure
PCA representation of transcriptomic comparison of Pl-MSCs, Mb-MSCs and BM-MSCs and ingenuity pathways analysis. (a) Mb-MSCs (red) and Pl-MSCs (green) are overlapping clearly indicating the impossibility to differentiate these MSCs at a transcriptional level. They can clearly be differentiated from bone marrow MSCs (blue). (b) The PCA overlapping between every cell sorted sub-population from membrane (CD29+ CD90+ red; CD29− CD90+ blue; CD90− CD29+ green) or nonsorted cells (Mb-MSCs purple) indicate the impossibility to discriminate the different MSCs subpopulation at a transcriptional level. (c) PCA of different MSC subpopulation from placenta (CD29+ CD90+ red; CD29− CD90+ blue) or directly isolated cells (Pl-MSCs blue) indicate the impossibility to discriminate these MSCs subpopulation at a transcriptional level. (d) Ingenuity pathway analysis was able to define enriched pathways implicated in embryonic morphogenesis and organ development in fetal MSCs compared to BM-MSCs. (e) Ingenuity pathway analysis using organ formation and osteoblast differentiation molecular network shows an enrichment in proosteogenic genes in fetal MSCs compared to BM-MSCs.
When compared to BM-MSCs, 145 genes were significantly upregulated and 267 genes were downregulated in Mb-MSCs compared to BM-MSCs (Supplementary Table 1 and Supplementary Figure 5). Similarly, 154 genes were significantly upregulated (133 overlapping with Mb-MSCs upregulated genes) and 272 genes were downregulated (238 overlapping with Mb-MSCs downregulated genes) in the Pl-MSCs compared to BM-MSCs (Supplementary Table 2 and Supplementary Figure 5).
By ingenuity and David analysis, we were able to define several pathways and genes implicated in embryonic morphogenesis and organ development upregulated in the Pl and Mb-MSC compared to BM-MSC (Figure
We then performed ingenuity pathway analysis building organ formation and osteoblast differentiation molecular networks. We found 14 genes upregulated in Pl/Mb MSC implicated in osteogenic differentiation in literature such as BMP, IGFBP4, IL6, HGF, and PTGS2 (Figure
Amniotic-membrane-derived MSCs (Mb-MSCs) were used for this part of the study as they are similar to the MSCs derived from the placenta and more abundant. In our cell culture and differentiation settings, the Mb/Pl MSCs displayed no differences in their ability to differentiate toward osteoblasts compare to BM-MSCs. OA treatment increased the differentiation for both Mb-MSC and BM-MSC at days 14 and 21 as demonstrated by Alizarin red staining, with Mb-MSCs displaying significantly increased osteogenic differentiation (Figures
Osteoactivin trigger increased osteogenic differentiation in human fetal and bone marrow MSCs. (a) Illustration of increased osteogenic differentiation with OA compared to control (without OA) after 21 days in Mb-MSCs and BM-MSCs. (b) Quantification of osteogenic differentiation with or without Osteoactivin (**
We analyzed the phosphorylation pattern of a range of phosphokinase after 4 h of OA stimulation in Pl-MSCs and BM-MSCs (Figures
Phosphokinase array analysis 4 h after osteoactivin stimulation. After 4 h of OA stimulation, cells were harvested and protein extracts analyzed with the human Phosphokinase array from R&D system. (a) Phosphokinase profile in Pl-MSCs and BM-MSCs using the proteome profiler. (b) Quantification of phosphokinase optical density. Measurements were obtained with image J software (NIH). ERK2 is activated in placental MSCs when submitted to osteoactivin stimulation.
MSCs are thought to have great therapeutic potential due to their capacity for self-renewal and multilineage differentiation [
Currently, the bone marrow is perceived as the major source of MSCs for cell therapy. However, aspiration of BM involves invasive procedures. The frequency, differentiation, and growth potential of BM-MSCs decrease significantly with age [
In this study, we have isolated MSCs from placenta and fetal membrane using very simple isolation technique with the same great purity yield (more than 95%) than initial FACS sorting methods [
Currently, bone morphogenetic protein-2 and -7 (BMP-2 and -7) are the only biologic modifiers that have received the United States Food and Drug Administration (US-FDA) approval for clinical applications in orthopedic surgery. The BMPs low biologic activity is demonstrated by the doses of tens of milligrams of commercial BMP-2- and -7-containing products, whereas BMPs concentrations
Noteworthy, we have characterized the mesenchymal stem cells by the criteria used by the ISCT [
We recently demonstrated that OA acts downstream of BMP-2, and our results indicate that OA may have similar osteoinductive effects to BMP-2 in mice.
We investigated the response of fetal MSCs to OA as compared to BM-MSCs. We demonstrate that OA can induce osteogenic differentiation in human MSCs. More interestingly, fetal-derived MSCs display better response to OA than BM-MSCs. We finally demonstrate that OA can also be used as a complement for osteogenic-induced differentiation with fetal MSCs. Finally, in accordance with the literature, we document that the induction of osteogenic differentiation following OA stimulation involved ERK1/2 pathway activation.
Considering that MSCs are way more abundant in membrane compared to placenta, we, therefore, stand that fetal membranes could be used to build MSCs banking program in order to meet clinical threshold in bone fracture reparation. Isolation of Mb-MSCs through selective culture in DMEM-low glucose supplemented with 20% serum and antibiotics seems to be the most efficient process. This process is very adapted for automation compatible with large cell banking programs.
Moreover, the increased capacity of response to osteogenic differentiation upon osteoactivin treatment prompts us to study the role of fetal membrane MSCs in experimental preclinical model of bone regeneration. Indeed, critical animal studies would be necessary to determine how MSCs are recruited and survive at the fracture site, their repair effectiveness, and the mechanisms through which they exert their actions.
This work was funded by Qatar Foundation NPRP Grant 08-663—3-140 and NPRP 09-1099-3-279 Qatar Foundation UREP Grant 06-116-1-023, Qatar Foundation, Qatar Technology Transfer Grant.