Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.
Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.
Mesenchymal stem/stromal cells (MSC) are emerging as a promising candidate for use in cell-based therapies 1. Most applications appear to target MSC-mediated tissue repair or immune regulation 2. In many of these applications, allogeneic MSC may be as effective as autologous MSC 3. The use of allogeneic MSC has the economic advantage of being compatible with the large-scale manufacture of multiple cell doses from a single source tissue 4 to treat many patients.
Historically, pre-clinical and clinical studies have utilized MSC-derived from the bone marrow 4. Bone marrow is generally collected from the iliac crest of a volunteer donor. This process is invasive, and only a small volume of marrow (~20 ml) is collected through a single puncture. Generating clinically meaningful numbers of MSC requires extensive in vitro expansion. Cell potency decreases with passage number 3, creating a paradox where the theoretical number of cells required for clinical efficacy is increased the more the cell population is expanded. In contrast to bone marrow aspirates, term placenta is a physically large starting tissue (typically 500-750 g 4), which can be harvested aseptically during cesarean section with no risk to the donor. MSC derived from placenta have long-term proliferation 5 and immunomodulatory capacity 6, superior to bone marrow-derived MSC. In a previous study, we demonstrated that a single term placenta contained sufficient MSC for the manufacture of up to 7,000 clinical doses 4. These characteristics make placenta an ideal source tissue for the manufacture of allogeneic MSC.
The placenta is a fetomaternal organ consisting of both fetal and maternal tissue 7, and thus MSC of fetal or maternal origin can be, theoretically, isolated. The following references provide detailed information on the development and pathology, as well as microscopic and macroscopic examination of the human placenta and adnexa 8,9. The placenta proper is comprised largely of fetal blood vessels and secretory and supporting cells called trophoblasts, making up the chorionic villi covered by the chorion frondosum (plate) 8. The branched placental villi are bathed in maternal blood delivered from the uterine spiral arteries, enabling nutrient, hormone and gas exchange between fetus and mother. The placenta is anchored to the endometrium via maternal decidual stromal cells and fetal extravillious trophoblasts are interspersed in extracellular matrix 8. The villi converge onto the fetal chorionic plate where they form the umbilical cord 8.
An outcome of the first International Workshop on Placental-Derived Stem Cells (2008) was an appreciation of the need to standardize the isolation and characterization of cells from human term placenta 10. Because of the anatomy of the placenta, dissection of the different tissues, isolation of MSC and anticipated culture outcomes can be overwhelming for newcomers to the field. In this protocol, the harvest of placental chorionic tissues, followed by MSC isolation and expansion is thoroughly detailed. MSC characterization via flow cytometry and in vitro differentiation are considered routine 5,11-13, and thus only briefly detailed here.
As highlighted in a recent systematic literature review 14, MSC obtained from the placental chorionic villi are generally assumed to be fetal. Although, only 18% of studies examined the origin of the MSC obtained, and of those, only half of the studies reported fetal MSC and the other half reported maternal or mixed MSC populations. Each of the three tissue components described herein (chorionic villi, chorionic plate and decidua basalis) are composed primarily of the fetal membrane/villi, and a small proportion of uterine-derived maternal cells, which remain attached to the delivered placenta. We provide data demonstrating that isolating MSC from the maternal side of the placenta, rather than the fetal side of the placenta, as we have previously reported 5,11, is a more appropriate starting material if maternal MSC are desired. This protocol also describes the use of XY FISH to validate fetal or maternal contribution to cell cultures. While this is a standard protocol from the manufacturer, this analysis is often neglected and its importance underestimated 14.
The Human Research Ethics Committees at Mater Health Services, Royal Brisbane and Women's Hospital, Queensland University of Technology and the University of Queensland approved the research and collection of human placenta samples used in the study. All protocols complied with national research guidelines. Patients provided informed written consent for the use of tissue for research purposes.
Third trimester placentas were obtained from healthy mothers following routine Caesarean section (CS) births at term from the above-mentioned hospitals in Brisbane, Australia. Male discordant pregnancies for term samples were utilized in this study to distinguish fetal from maternal cells. Fetal gender had been determined by ultrasound prior to birth and/or visual inspection of the neonate at birth by clinical staff. X and Y chromosome fluorescence in situ hybridization (FISH) was utilized to further validate gender and fetal or maternal origin of tissue preserved from the original placentas.
1. Prior to Harvest, Prepare the Following
Note: When making up enzyme solutions, the specific requirements and concentrations provided by the manufacturer for each product should be followed. Enzymes are often provided as a crude mix of proteins, therefore similar enzymes from different companies are likely to have different activities/concentrations. For this reason the manufacturer's advice should be acknowledged and solution preparation may have to be modified accordingly.
2. Isolation of the Placental MSC
3. Subculture of pMSCs
The placental MSC isolation procedure is summarized in Figure 1. The three areas of the placental anatomy from which MSC were isolated are highlighted in Figure 2. These are the maternal decidua, as well as the largely fetal tissues of the chorionic plate and chorionic villi. Many text books, articles and online resources detail the development and functional role of the various placental tissues (please see reference 8).
Morphology of cultures 48 hr after isolation and removal of the tissue debris.
Following 48 hr of culture, the MSCs will have attached to the tissue culture plastic, while RBCs and most other cellular debris will not have attached. At this time, the medium must be replaced with 35 ml of fresh culture medium. Prior to this medium exchange, it is difficult to make precise observations because of the large numbers of RBCs, which will obstruct visual assessment. The appearance of the culture supernatant may vary substantially between placenta donors. This variation can be seen visually in Example 1 and 2 (Figure 3A). These two MSC isolations, which appeared to be very different, were performed simultaneously from two different placentas. However, once washed, both cultures appeared similar (see washed example 3, Figure 3A).
Under a microscope, after the 48 hr media exchange, only a few cells will be attached to the flask (Figure 3B), and the cells will appear significantly different from more extensively expanded MSCs. Some debris and RBCs will be visible as floating or attached clumps and these will not interfere with the growth of the MSCs. The longer fibroblastic cells adhered to the bottom of the flask are likely to be a mix of cells including MSC, hematopoietic, trophoblastic or endothelial cells. Again, the non-MSC cells do not compromise the MSC cultures, as these cells will not generally survive more than 1-2 passages in the MSC culture conditions. Despite some variation in the initial appearance of cultures, the subsequent expansion results are generally consistent.
Morphology of MSC cultures over time.
In this representative example, seven days following isolation, small fibroblastic MSC colonies were visible, although non-MSC cells could also be seen as round or loosely attached cells (Figure 4A). The attached cells are what was originally termed a "colony unit forming-fibroblast" (CFU-F) 17 and later termed MSC 18. Thirteen days following isolation, fibroblastic MSC colonies were large (Figure 4B). Generally, the monolayer will be 80-90% confluent at this point and the cells should be passaged. From passage 2 onwards, the placental MSC monolayer will develop the characteristic whirlpool-like morphology at confluence (Figure 4C). Unless the cells are passaged at low density, the CFU-F formation will no longer be observed. At low density, placental-derived MSC have a smaller, squarer appearance than adult bone marrow-derived MSC 1. While placental-derived MSC and bone marrow-derived MSC exhibit similar proliferation rates, the placental-derived cells are less prone to rapid senescence 5.
Characterization of placental MSC in vitro.
Each expanded cell population must be characterized to ensure that it conforms to standard MSC criteria 16, including (1) plastic adherence (2) the presence of mesenchymal surface markers and the absence of hematopoietic surface markers, and (3) the capacity to undergo mesodermal differentiation.
Placental MSCs are plastic adherent.
As shown in Figure 4, the MSCs in culture are plastic adherent and have a fibroblastic-like morphology; this validates that the cells meet the first criteria that define MSC 16.
Placental MSC display mesenchymal surface markers.
The second defining MSC characteristic is the presence of mesenchymal surface markers and the absence of hematopoietic surface markers 16. As there is no single marker capable of definitively identifying an MSC, panels of markers are generally utilized in conjunction with flow cytometry analysis to identify cells that are mesenchymal, but not hematopoietic. In the representative data set provided here (Figure 5A), we evaluated cell expression of mesenchymal markers CD73, CD105, CD90, CD146, and CD44, the hematopoietic markers CD45 and CD34, and HLA-DR, as well as the endothelial marker CD31. All of the antibodies utilized in this placental MSC characterization process are listed in the Table of Materials/Equipment. Staining was performed as per the manufactures instructions, with analysis methods described here 19.
The cells were positive for the mesenchymal markers CD73, CD105, CD44, and negative for the cell surface markers CD45, CD34, HLA-DR and CD31, as expected 5,20. Approximately, 37% and 57% of cells in our representative data set were positive for CD90 and CD146 21, respectively. Both CD90 and CD146 are commonly utilized MSC markers 21. MSC cell surface marker profiles may be different depending on MSC tissue source, medium composition, or passage number 22. In our many years of experience, we have not observed long-term contamination of placental-derived MSC with non-mesenchymal cells following 1-2 passages 5,11.
Placental MSC display mesenchymal differentiation potential
By definition, MSC must possess in vitro mesodermal differentiation capacity 5,13. Mesodermal differentiation potential is commonly assessed through either tri- or bi-lineage differentiation assays. Bi-lineage assays generally assess osteogenic and adipogenic differentiation capacity, while tri-lineage assays additionally assess chondrogenic differentiation capacity. In the representative results presented here, we show that the expanded MSC populations form both calcium deposits, indicative of osteogenic differentiation, and lipid vacuoles, indicative of adipogenesis (Figure 5B).
To characterize the MSC populations reported here, we seeded cells into 24 culture wells at 6 x 104 cells in 1 ml of induction medium. The medium components are listed in the Table of Materials/Equipment. While induction medium formulations are common in the literature, there is considerable variability in published formulations. For this reason we briefly list our induction medium formulations and staining approaches here. Osteogenic induction medium contained DMEM-HG, 10% FBS, 1x antibiotic antimycotic solution, 10 mM β-glycerol phophate, 100 nM dexamethasone, and 50 μM L-ascorbic acid 2-phosphate. Adipogenic induction medium contained DMEM-HG, 10% FBS, 1x antibiotic antimycotic solution, 10 μg/ml insulin, 100 nM dexamethasone, 200 μM indomethacin, and 500 μM 3-isobutyl-1-methyl xanthine. Here, cultures were maintained in a 37 °C, 5% CO2 incubator and cultured for 14 days. Induction medium was exchanged twice per week over the culture period. Following 14 days of induction, cultures were characterized for either bone-like matrix (osteogenesis) or lipid vacuoles (adipogenesis) as per our previous publication 5. Osteogenic calcium matrix deposition analysis was achieved by first aspirating off the medium, fixing the cultures with 4% paraformaldehyde for 20 min, washing the monolayer with DPBS, and then staining with Alizarin Red S according to the manufactures instructions. Adipogenic induction was assessed by aspirating off the medium, fixing the cultures with 4% paraformaldehyde for 20 min, washing the monolayer, and staining with Oil Red O solution according to the manufactures instructions. Stained calcium deposits and oil vacuoles were then visualized with a light microscope, and images saved for future reference.
In previous publications, we have characterized the differentiation potential of placental-derived MSC more extensively 4,5. Placental-derived MSC osteogenesis is similar to bone marrow-derived MSC, while adipogenesis is generally less efficient in placental-derived MSC 5. We do not routinely carry out chondrogenic differentiation for several reasons, although this has been previously reported by us for placental-MSC 5. Firstly, while mesodermal differentiation capacity is a defining characteristic of MSC, it is likely of secondary importance 23-25, especially where the therapeutic benefit is likely to be derived from the MSC paracrine secretions 26. Secondly, although Dominici et al. proposed minimal criteria for the clinical production of human adult bone marrow-derived MSC 16, more recent studies indicate MSC from different niches have different inherent properties and differentiation capabilities 5,13,27-32. In fact, Parolini et al. proposed that placenta-derived MSC should differentiate into "one or more mesodermal" lineages rather than all three lineages 10. Finally, many MSC studies exclude chondrogenic differentiation as it occurs through a similar intracellular signaling pathway as osteogenesis (TGFβ family pathway) 33-35.
Placental MSC are maternal in origin using this method of culture, despite the anatomical location of the starting material.
Many publications assume that cells isolated from the fetal chorion yield fetal MSC upon culture 14. However, as we have reported previously 5, all cultures derived from fetal chorion, using this protocol, rapidly become enriched for maternal MSC as would intuitively be expected for the maternal decidual MSC cultures. In these representative results we used placental tissue from male babies so that it was possible to easily delineate the fetal and maternal cell contribution in the expanded cell populations. For these studies we used the XY FISH kit listed in the Table of Materials/Equipment, and followed the manufacturers instructions.
In the representative data provided here, the cultures derived from maternal decidua were ~90% maternal cells (XX) and ~10% fetal cells (XY) at passage 0 (Figure 6). By passage 2, the cell populations derived from the maternal tissues were ~100% maternal cells (XX), and fetal cells (XY) were undetectable. This suggests that physical dissection of maternal tissue led to the enrichment of maternal cells in the subsequent cultures. However, it is critical to consider the culture outcomes from the fetal chorionic villi and chorionic plate-derived cultures. At passage 0, both fetal chorionic villi and chorionic plate derived cultures were ~85% XY, or of fetal origin, indicating that targeted dissection enriched for fetal cells (Figure 6). At passage 0, both cultures contained ~15% maternal cell (XX) contamination. Surprisingly, at passage 2, both fetal cultures were populated with ~100% maternal cells (XX), and fetal cells (XY) were no longer detectable. XY FISH analysis reveals that maternal cells (XX chromosomes) rapidly and consistently takeover the cultures derived from the fetal chorionic tissues. This is a critical culture observation that is often overlooked 5. The detail of this analysis is included in this protocol because it demonstrates the very important observation that maternal cells rapidly populate all cultures when DMEM supplemented with 10% FBS is used without additional factors designed to support the fetal-derived populations.
Figure 1: Summary of placental MSC isolation procedure. (Step 1) Orientate yourself with the placenta anatomy. (Step 2) Manually dissect 10 g of tissue from either the decidua, chorionic villi or the chorionic plate using scissors. (Step 3) Mince the dissected portions of decidua, chorionic villi or the chorionic plate tissues into fine pieces with scissors or a scalpel.
(Step 4) Liberate cells from the fine pieces via a 1-2 hr digestion in dispase and collagenase I.
(Step 5) Separate the cells from the fibrous tissue by pulse centrifugation and/or washing them through a cell strainer. Collect and resuspend cells in culture medium and placed into culture flasks. Please click here to view a larger version of this figure.
Mesenchymal stromal cells (MSC) will be selected based on their propensity for plastic adherence and capacity to survive and proliferate in the culture medium. Finally, in the results section, the expanded MSC can be characterized and stored for use in future experiments.
Figure 2: Anatomy of the human term placenta and tissues isolated in this procedure. The first tissue to be harvested is maternal decidua. Decidua is tissue that remains as a thin layer on the surface of the placenta after it is shed from the uterine wall (decidua is identified by the green markers). The second tissue that will be harvested from the interior of the placenta is the fetal chorionic villi (blue markers). The third tissue to be harvested is fetal chorionic plate (red markers) (adapted from reference 36). Please click here to view a larger version of this figure.
Figure 3: Morphology of cultures 48 hr after isolation and removing the tissue debris. (A) The appearance of the culture supernatant can vary substantially between placenta donors before washing off the debris. Example cultures 1 and 2 demonstrate this variation. These two isolations were performed simultaneously, but from two different placentas. Once washed cultures will be clear of RBCs and tissue debris as shown in example 3. The subsequent expansion results are generally consistent. (B) Following the 48 hr media exchange, only a few cells will be attached to the flask. Scale bar = 200 μm. Please click here to view a larger version of this figure.
Figure 4: Morphology of MSC cultures over time. (A) Seven days after isolation, small fibroblastic MSC colonies are visible although non-MSC cells will also be present as round or loosely attached cells. (B) 13 days after isolation, fibroblastic MSC colonies are large and often the monolayer of MSC is confluent and ready to passage. (C) From passage 2 onwards, the MSC monolayer will develop a characteristic whirlpool-like morphology at confluence. Scale bar = 200 μm. Please click here to view a larger version of this figure.
Figure 5: MSC characterization by flow cytometry and mesodermal differentiation. (A) The placental chorionic villi-derived MSC display a classic MSC marker profile by flow cytometry analysis, although for these cell surface markers, expression is similar for all types of human MSC. Each histogram shows the signal intensity (x-axis) versus the normalized cell count on the y-axis (% of Max). In this representative data set the cells were positive for CD73, CD105 and CD44, and negative for the hematopoietic markers CD45 and CD34 and HLA-DR. (B) Placental MSC generally undergo robust osteogenic differentiation, however (C) adipogenic differentiation can be less efficient than with bone marrow-derived MSC. Images in captions B and C were taken at 40X magnification. Please click here to view a larger version of this figure.
Figure 6: Placental MSC are maternal in origin using this method of culture, despite the anatomical location of the starting material. The plots show quantification of the fetal (male, XY) and maternal (female, XX) cell composition of the placental MSC cultures isolated from decidual, chorionic villi and chorionic plate tissues at every second passage. Maternal cells (XX) rapidly and reproducibly take over the cultures derived from the fetal chorionic tissues. Fetal = male = XY chromosomes detected in an individual cell, maternal = female = XX chromosomes detected in an individual cell. Data presented here was from N = 3 independent donor placentas from male babies, with a minimum of 100 cells stained for XY FISH and counted for each data point. Bars represent averages, and error bars reflect one standard deviation. Please click here to view a larger version of this figure.
The placenta is a physically large fetomaternal organ, from which fetal or maternal MSC can be isolated 22. Herein, we provided a detailed overview of placental anatomy and instruction on how to specifically dissect decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue (Step 2.2-2.4). Subsequently, we outlined a robust protocol that enables MSC isolation from each of these three tissues (Step 2.5-2.6). Placental MSC expansion is efficient and cultures appear similar to bone marrow-derived MSC cultures (Figures 3 and 4). Osteogenic differentiation is reliable (Figure 5B), while adipogenic differentiation is generally less efficient (Figure 5C) 5.
Many newcomers to the field will presume that cultures derived from fetal chorionic villi or chorionic plate tissues will be enriched for fetal MSC. However, in our hands fetal cell enrichment is only transient when standard MSC expansion medium such as DMEM-LG + 10% FBS is utilized 5. Here we provide representative results using placental tissues derived from a male baby. By using placenta tissue from a male baby, the fetal cells are readily identifiably as having XY chromosomes, while maternal cells are identifiable as having XX chromosomes. Figure 6 shows XY FISH results for a representative culture. While fetal MSC (XY) are enriched (up to 80%) in the initial cultures derived from fetal chorionic villi or chorionic plate tissues, these same cultures are rapidly overtaken (~100%) by maternal (XX) MSC over the first two passages. In standard medium, composed of DMEM-LG + 10% FBS, the few maternal-derived MSC that contaminate the fetal tissues outcompete the fetal-derived cells in culture.
A critical step outlined in this protocol is an appreciation of the placental anatomy and from where fetal and maternal tissue can be most effectively harvested. As outlined in the representative results section, dissection of fetal tissue does enable transient enrichment for fetal-derived MSC. Improvements in expansion medium formulation, through specific exogenous growth factor medium supplementation should allow selective expansion of the fetal-derived MSC populations, and manufacture a cell product that is enriched for fetal rather than maternal cells (our group is currently developing such medium formulations). The manufacture of fetal MSC populations may have a number of advantages, as fetal MSC are purported to have greater angiogenesis and immunosuppressive properties than equivalent maternal MSC populations 37.
In each of the described isolation protocols, we used approximately 10 g of tissue. A whole placenta is typically 500-750 g, and in previous work we demonstrated that through automated tissue digest and a cell expansion bioreactor processes that it should be possible to manufacture over 7,000 clinical cell doses from a single placenta 4. These numbers highlight the potential suitability of placental-derived MSC in allogeneic MSC therapies, and the significance of this method regardless of MSC origin (fetal or maternal). From a therapeutic perspective, it is most critical that users have a full understanding of the cell product and the capacity to reliably manufacture this cell product. We hope that our video will assist researchers to understand placenta anatomy, isolate MSC from placenta, and anticipate the likely fetal or maternal cell composition of their cultures.
The authors have nothing to disclose.
RP was supported by a National Health and Medical Research Council (NHMRC) Postdoctoral Training Fellowship. VS was supported by a University of Queensland International Postgraduate Student scholarship. MRD was supported by the NHMRC and Inner Wheel Australia.
We thank clinical and nursing staff for assisting in patient consent and sample collection. We thank Prof. Nickolas Fisk, Prof. Kerry Atkinson and Dr Rohan Lourie for insightful discussions in obstetrics, feto-placental development and placenta anatomy.
Reagents | |||
HBSS | Gibco/Invitrogen | 14185-052 | Long name: Hanks Balanced Salt Solution |
FBS | Gibco/Invitrogen | Long name: Fetal Bovine Serum | |
trypsin-substitute | Gibco/Invitrogen | 12563-029 | Long name: TrypLE Select |
DMEM-LG | Gibco/Invitrogen | 11885-092 | Long name: Dulbecco’s Modified Eagles Medium-Low Glucose |
DMEM-HG | Gibco/Invitrogen | 11965118 | Long name: Dulbecco’s Modified Eagles Medium-HIgh Glucose (4.5 g/L) |
PBS (Mg+Ca+ free) | Gibco/Invitrogen | 14190-250 | Long name: Dulbecco's Phosphate buffered saline, magnesium and calcium free |
Anti- anti- | Gibco/Invitrogen | 15240-062 | Long name: antibiotic-antimycotic solution x100 |
paraformaldehyde powder or 4% solution | any | ||
Collagenase I | Invitrogen | 17100-017 | 2500U/ml |
Dnase I | Sigma | D5025 | 10 mg/mL in 0.15 M NaCl |
Dispase | Invitrogen | 17105-041 | 10 mg/ml in water (20,000 U/ml). |
Material | Company | Catalog Number | Comments |
Disposables: | |||
50 ml centrifuge tubes | Falcon or any brand | ||
petri dishes, sterile plactic (25 cm and 10 cm diameter) | Nunc | ||
Cell strainers (100 micron) | Becton Dickinson | 352340 | |
175 and 75 cm2 (T175 and T75) tissue culture flasks | Nunc | ||
5 ml, 10 ml, 25 ml sterile serolgical pipettes | any brand | ||
Material | Company | Catalog Number | Comments |
Equipment: | |||
Centrifuge | |||
tissue culture incubator 37 degrees celcius, 5 % CO2 | |||
Biological safety cabinet | |||
Sterile scissors and tweezers | |||
Tube racks | |||
Pipette-boy or equivalent | |||
Gilson type pipetters and sterile tips 1000 µl, 200 µl, 20 µl. | |||
rocking or shaking incubator (37 degrees celcius) | |||
personal protective equipment | |||
cleaning solutions (suitable for blood) | |||
waste containers, correct disposal bins for tissue/blood | |||
Reagents for MSC characterization | |||
Antibody (Clone ID) | Manufacturer | Catalogue No. | Isotype |
CD73 (AD2) | Miltenyi Biotec | 130-095-183 | Mouse IgG1 |
CD105 (43A4E1) | Miltenyi Biotec | 130-094-941 | Mouse IgG1 |
CD90/Thy-1 (AC122) | Miltenyi Biotec | 130-095-403 | Mouse IgG1 |
CD45 (5B1) | Miltenyi Biotec | 130-080-202 | Mouse IgG2a |
CD34 (AC136) | Miltenyi Biotec | 130-090-954 | Mouse IgG2a |
HLA-DR (AC122) | Miltenyi Biotec | 130-095-298 | Mouse IgG2a |
CD31 (PECAM-1) | BD Pharmingen | 555446 | Mouse IgG1 |
CD146 (541-10B2) | Miltenyi Biotec | 130-092-849 | Mouse IgG1 |
CD44 (DB105) | Miltenyi Biotec | 130-095-180 | Mouse IgG1 |
Isotype Controls (Clone ID) | Manufacturer | Catalogue No. | |
Mouse IgG1 Isotype (IS5-21F5) | Miltenyi Biotec | 130-092-214 | |
Mouse IgG1 Isotype (IS5-21F5) | Miltenyi Biotec | 130-092-212 | |
Mouse IgG1 Isotype (IS5-21F5) | Miltenyi Biotec | 130-092-213 | |
Mouse IgG2a (S43.10) | Miltenyi Biotec | 130-091-837 | |
Mouse IgG2a (S43.10) | Miltenyi Biotec | 130-098-849 | |
MACS Buffer | Miltenyi Biotec | 130-091-221 | |
Osteogenic differentation | Manufacturer | Catalogue No. | |
DMEM-HG | Gibco/Invitrogen | 11965118 | Long name: Dulbecco’s Modified Eagles Medium-HIgh Glucose (4.5 g/L) |
FBS | Gibco/Invitrogen | Long name: Fetal Bovine Serum | |
Anti- anti- | Gibco/Invitrogen | 15240-062 | Long name: antibiotic-antimycotic solution x100 |
Dexamethasone | Sigma | D4902 | |
β-Glycerol Phophate | Sigma | 50020 | |
L-ascorbic acid 2-phosphate | Sigma | A8960-5G | |
Alizarin Red S | Sigma | A5533-25G | For calcium matrix staining |
Adipogenic differentation | Manufacturer | Catalogue No. | |
DMEM-HG | Gibco/Invitrogen | 11965118 | Long name: Dulbecco’s Modified Eagles Medium-HIgh Glucose (4.5 g/L) |
FBS | Gibco/Invitrogen | Long name: Fetal Bovine Serum | |
Anti- anti- | Gibco/Invitrogen | 15240-062 | Long name: antibiotic-antimycotic solution x100 |
insulin | Sigma | I2643 | |
Dexamethasone | Sigma | D4902 | |
Indomethacin | Sigma | I7378 | |
3-isobutyl-1-methyl xanthine | Sigma | I5879 | |
Oil Red O solution | Sigma | O1391-250ML | For lipid vacuole staining |
XY FISH kit to determine fetal or maternal origin of cells | |||
XY chomosome FISH kit | Vysis (Abbott Molecular) | 07J20-050 | Long name: CEP X SpectrumOrange/Y SpectrumGreen Direct Labeled Fluorescent DNA Probe Kit |