Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.
In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as “Mesangiogenic Progenitor Cells”.
Mesenchymal Stromal Cells (MSCs) are of relevant clinical value for their multi-lineage differentiation capacity and their ability to support hemopoiesis, to secrete growth factors/cytokines as well as to play a role in immunoregulation1. In the definition of MSC-based therapies cell production and application have been the object of extensive clinical and pre-clinical research2, with particular attention to specific international regulation for the safety and efficacy of cell based medicinal product (CBMP) treatments3. Human MSCs are extensively cultured in media containing supplements and reagents of animal origin, such as fetal bovine serum (FBS) and bovine trypsin. Therefore, alongside with infectious risks associated with cell manipulation, patients also face prion exposure as well as immunological risks linked to proteins, peptides or other biomolecules of animal origin that could persist after cell harvesting and transplantation4.
To circumvent the problem, we cultured human bone marrow (hBM)-derived MSCs in animal-free medium, replacing FBS with pooled human AB type serum (PhABS). Under these conditions, alongside growing MSCs we identified a novel cell population. These cells were morphologically and phenotypically different from MSCs and showed a distinctive gene expression profile as well as characteristic growing/adhesion properties. They retained both mesengenic and angiogenic potential and therefore were named Mesodermal Progenitor Cells (MPCs)5. Subsequently, we were able to define selective and reproducible culture conditions to generate MPCs at high grade of purity6.
We further investigated the morphological and biological properties of MPCs. The MPCs showed to be nestin-positive, slow in cycling, Ki-67-negative, and with chromosomes characterized by long telomeres5. They expressed pluripotency-associated transcription factors Oct-4 and Nanog rather than MSC master regulators Runx2 and Sox97. Phenotypically, MPCs expressed endoglin (CD105) at lower level than MSCs while lacking mesenchymal markers CD73, CD90, CD166. MPCs also showed a distinctive pattern of adhesion molecules characterized by consistent expression of PECAM (CD31), integrins αL (CD11a), αM (CD11b), αX (CD11c) as well as integrin β2 (CD18) that specifically sustains podosome-like structures8. In standard MSC expansion media, MPCs promptly differentiated into MSCs through an intermediate stage featuring the activation of Wnt5/Calmodulin cell signaling9. MPCs also retained angiogenic properties, as demonstrated by their ability to sprout from spheroids in murine extracellular matrix (ECM) protein 3D cultures. The angiogenic potential was rapidly lost after MPC differentiation along the mesengenic lineage.
Here we present protocols optimized to isolate and to characterize highly purified MPCs from hBM blood samples. Reproducible protocols for MPC mesengenic and angiogenic differentiation are also described.
NOTE: After written consent, hBM samples were obtained during orthopedic surgery for hip replacement. Immediately after femoral neck osteotomy and before femoral reaming a 20 ml syringe containing 500 U.I. of heparin, was used to aspirate fresh BM. The protocol is to be considered widely applicable to any BM source.
1. Isolation of Human Bone Marrow Mononuclear Cells (hBM-MNCs)
2. Isolation of MPCs from hBM-MNCs
3. Cell Characterization
4. Mesengenic Differentiation of MPCs
5. MPC Spheroid Sprouting Assay
The selective culture conditions described here have allowed the isolation of a novel adherent and almost monomorphic cell population as 1.0% of the hBM-MNCs (0.5 – 2.0 x 106 hBM-MNCs from 5 – 10 ml of fresh BM samples)5,6. We identified these large (40 – 60 µm in diameter), rounded, quiescent, Ki-67-negative cells as MPCs5. Morphologically, they are characterized by a distinctive fried egg-shape with a thick core region surrounded by a flat thin periphery showing lots of filopodia at higher magnification power (white arrows in Figure 1 A.1). Polar elongation of the outer cell boundary is often observed (black arrows in Figure 1 A.1). Such morphology is clearly different from the typical spindle-shaped mesenchymal stromal cell appearance reported in standard MSC cultures. Flow cytometry showed over 95% of freshly isolated MPCs to express CD31 and CD45 while mesenchymal associated markers CD90 and CD7313 were undetectable (Figure 1 A.2). We regard this restricted set of four antigens as indicative for MPCs. Further distinguishing features of MPCs are dotted F-actin distribution revealing a number of podosome-like structures (red in Figure 1 A.3) and intense expression of nestin (green in Figure 1 A.3), which is not detected in the cells stained with isotypic control antibody (data not shown).
Culturing MPCs in standard RS medium designed for MSC expansion results in rapid differentiation into exponentially growing MSC-like cells (Figure 1 B.1). After two passages cells finally switch their phenotype from CD73negCD90negCD45+CD31+ to CD73+CD90+CD45negCD31neg (Figure 1 B.2). In the process, MPCs re-organize F-actin into stress fibers while nestin expression becomes confined to few rare cells (Figure 1 B.3). MPC mesengenic differentiation into a definite MSC-like phenotype occurs through two distinct steps revealed by different cell morphologies. After one week in MSC RS medium a residual population of MPC-like cells is still detectable within a confluent layer of flat, polygonal multi-branched cells (P1-MSCs in Figure 2 A). A further passage is required to obtain an almost monomorphic culture of spindle-shaped MSC-like cells (P2-MSCs in Figure 2 A). These exponentially growing cells can readily differentiate into osteoblasts or adipocytes when transferred into selective media for at least 2 weeks, thus confirming their MSC nature. In osteogenic induced cultures, calcium deposits can be detected by either colorimetric Alizarin-S stain or specific fluorescent dyes (green in Figure 2 B). After adipogenic induction cells show lipid droplet accumulation as revealed by either colorimetric Oil Red or fluorescent Nile Red stain (red in Figure 2 B).
MPC typing was confirmed by sprouting angiogenesis assay. MPCs showed their ability to invade (over 50 µm) murine ECM protein gel from 3D spheroids after 24 hr VEGF-stimulus (Figure 2 C). After one week invading cells were detected at 300 – 600 µm distance. Conversely, invasion capacity was lost in P2-MSCs after mesengenic differentiation (Figure 2 D).
Figure 1. Freshly Isolated MPCs have Distinctive Features. Culturing hBM-MNCs in DMEM/10% PhABS for seven days gives rise to a population of quiescent MPCs (A) easily distinguishable from MSCs (B) in terms of morphology (A.1, B.1, scale bars = 100 µm), phenotype (A.2, B.2), F-actin distribution (red in A.3, B.3), and nestin expression (green in A.3, B.3, scale bars = 20 µm). Please click here to view a larger version of this figure.
Figure 2. MPCs Differentiate into Standard MSCs and Show Sprouting Angiogenesis in vitro. Replacement of DMEM/PhABS with commercially available RS medium designed for MSC expansion triggers mesengenic induction of MPCs. After one week in culture few residual MPCs are still detectable (P1-MSCs) while a further passage in MSC-RS medium leads to a population of confluent MSC-like cells (P2-MSCs, A scale bars = 100 µm). P2-MSCs terminally differentiate into osteocytes or adipocytes under proper stimuli as revealed by calcium deposition (green in B) and lipid droplet accumulation (red in B, scale bars = 200 µm), respectively. MPCs show consistent sprouting from spheroids in murine ECM protein gel (C) at a difference with P2-MSCs (D, scale bars = 1.0 mm). Please click here to view a larger version of this figure.
In the last decades, MSCs have been extensively researched and pre-clinically evaluated for possible application in the treatment of various bone/articular, immunological, neurological, cardiovascular, gastrointestinal and hematological disorders14,15. The easy and inexpensive isolation of multipotent MSCs, from many different tissues, together with their lack of significant immunogenicity16, contribute to make these cells one of the most interesting cell population to be applied in cell based therapies. Nonetheless, the very low frequency in the tissue of origin represents a great limitation to the MSCs application in clinics, forcing the expansion of these cells, in vitro, before the infusion or transplantation.
Expanded MSC cultures have revealed high grades of heterogeneity and variability17-19 making it difficult to reach a consensus about MSC production and characterization protocols. Moreover, recent investigations suggested the presence of multiple in vivo MSC ancestors in a wide range of tissues, which contribute to culture heterogeneity10,20. In fact, it has been proposed that particular culture conditions possibly select or simply promote specific sub-populations of MSCs progenitors present, in various percentages, in “crude” and unprocessed samples like bone marrow (hBM-MNCs) or adipose tissues (stromal vascular fraction)2. Thus, the variability in MSC-initiating cell populations together with the great number of different enrichment/isolation and culture protocols applied, represent a great obstacle to the definition of feasible MSC-based therapies.
A crucial factor affecting heterogeneity of MSC cultures is serum supplementation21. In our hands replacement of FBS with PhABS in primary cultures from hBM-MNCs, combined with high density seeding on hydrophobic plastics, led to the isolation of a novel highly adherent cell population with distinct biological features named MPCs5,6. We observed that the addition of small percentages of PhABS to FBS primary cultures also allowed MPC isolation, suggesting the presence of MPC inducing agents in the human serum6. At the moment, the MPC isolation/characterization protocol is a unique method available to obtain almost pure MPCs. The protocol has been carefully adjusted and it is highly reproducible for quality screening of MPC preparations before further applications.
MPCs could be used as a source for MSC production, thus limiting the variability introduced by use of unfractionated starting material. The precise definition of the multiple steps characterizing MPC mesengenic differentiation reported9 would allow synchronized mesenchymal cell expansion. Nonetheless, this latest condition could be realized exclusively applying highly purified MPC population, as a consequence the characterization of the cell products obtained by the protocol described here, results of crucial importance. This isolating method has been reported allowing MPC recovery with purity generally around 95%. However, donor/patient variability together with the variability related to the different batches of human pooled serum applied, could lead to a significant percentage of MSC-like cells co-isolated together with MPCs, under selective conditions.
It is not clear if these “contaminating” MSC-like cells could arise from the other different in vivo progenitors described in bone marrow22 or from uncontrolled and spontaneous MPC differentiation. In any case, a consistent percentage of MSC-like cells in the MPC products nullify the possibility to applying these cells as homogeneous starting material for the MSC expansion. Thus, here it has been suggested a simple and inexpensive method, based on the MPC resistance to trypsin digestion, increasing the purity of the MPC products. Similar or even better results in purifying MPC cultures could be achieved by fluorescent or magnetic cell sorting performing CD73 and/or CD90 depletion, but significantly prolonging the process time and increasing the costs.
Moreover, MPCs showed expression of pluripotency-associated markers and Nestin, all rapidly lost during mesengenic differentiation7. Sprouting assay revealed MPC ability to invade murine ECM protein gel. Taken together these results indicate that MPCs have to be considered a more immature progenitor, retaining angiogenic potential. Nonetheless, the initial enthusiasm about mesodermal differentiation potential of MPCs is actually waning. In fact, after more than 7 years of studies on MPCs, mesengenic and angiogenic potential have been extensively described5-9, but differentiation toward any other cells of mesodermal origin is still lacking. Thus, here we propose a new, and more rigorous, definition of these cells as “Mesangiogenic Progenitor Cells”, maintaining the acronym MPCs.
We also believe that most controversies about MSC angiogenic potential could be related to the heterogeneous composition of expanded cultures consisting of sub-populations of MPCs and MSCs in variable percentages23.
Finally, MPCs could also play a crucial role for the implementation of CBMPs applicable for tissue reconstruction, as these cells could also support the neo-vascularization. In fact, future studies on regeneration should take in consideration that the newly formed tissue growth should be supported by concomitant neo-angiogenesis. The co-existence of mesengenic and angiogenic potential in MPCs could significantly improve the regeneration potential of new therapeutic approaches that involve these interesting cells.
The authors have nothing to disclose.
The Authors would especially like to thank Dr. Paolo Parchi, department of Surgical, Medical and Molecular Pathology and Critical Care Medicine, University of Pisa, for providing bone marrow samples and his expertise in human osteo-progenitors
Matrigel Basement Membrane Matrix | BD Bioscience (San Jose, CA-USA) | 354230 | Murine ECM proteins Stock Concentration: 100% (9-12 mg/ml) Final Concentration: 100% |
Dulbecco's Phosphate-Buffered Saline (D-PBS) | Sigma (St. Louis, MO, USA) | D8537 | |
70 μm Filters | Miltenyi Biotec (BergischGladbach, Germany) | 130-095-823 | |
Ficoll-Paque PREMIUM | GE Healthcare (Uppsala, Sweden) | 17-5442-03 | medium for discontinuos density gradient centrifugation |
Pooled human AB type serum (PhABS) | LONZA (Walkersville MD-USA) | 14-490E | Final Concentration: 10% |
Glutamax-I | ThermoFisher (Waltham, MA USA) | 35050-038 | Stabilized L-Glutamine Stock Concentration: 100X Final Concentration: 2 mM |
Bovine Serum Albumin (BSA) | Sigma (St. Louis, MO, USA) | A8412 | Stock Concentration: 7.5% Final Concentration: 0.5% |
Sodium Azide | Sigma (St. Louis, MO, USA) | S8032 | Final Concentration: 0.02% |
Penicillin/Streptomycin (Pen Strep) | Gibco (Grand Island, NY, USA) | 15070-063 | Antibiotics Stock Concentration: 5,000 UI/mL penicillin, 5,000 ug/mL Streptomycin Final Concentration: 50 UI/mL penicillin, 50 ug/mL Streptomycin |
T-75 culture flask for suspension cultures | Greiner Bio-one (Frickenhausen, Germany) | 658 190 | |
T-75 culture flask TC treated | Greiner Bio-one (Frickenhausen, Germany) | 658170 | |
TrypLE Select | ThermoFisher (Waltham, MA USA) | 12563-011 | Animal- free proteases detaching solution Stock Concentration: 1X Final Concentration: 1X |
Trypsin/EDTA | ThermoFisher (Waltham, MA USA) | 15400-054 | Phenol red free Stock Concentration: 0.5% Final Concentration: 0.25% |
anti-CD90 APC antibody (CD90) | MiltenyiBiotec (BergischGladbach, Germany) | 130-095-402 | Final Concentration: 1:40 |
anti-CD45 APC-Vio770 antibody (CD45) | MiltenyiBiotec (BergischGladbach, Germany) | 130-096-609 | Final Concentration: 1:40 |
anti-CD73 PE antibody (CD73) | MiltenyiBiotec (BergischGladbach, Germany) | 130-095-182 | Final Concentration: 1:40 |
anti-CD31 PE Vio-770 antibody (CD31) | MiltenyiBiotec (BergischGladbach, Germany) | 130-105-260 | Final Concentration: 1:40 |
Mouse IgG1 APC antibody | MiltenyiBiotec (BergischGladbach, Germany) | 130-098-846 | Final Concentration: 1:40 |
Mouse IgG2a APC Vio770 antibody | MiltenyiBiotec (BergischGladbach, Germany) | 130-096-637 | Final Concentration: 1:40 |
Mouse IgG1 PE antibody | MiltenyiBiotec (BergischGladbach, Germany) | 130-098-845 | Final Concentration: 1:40 |
Mouse IgG1 PE Vio-770 antibody | MiltenyiBiotec (BergischGladbach, Germany) | 130-098-563 | Final Concentration: 1:40 |
Low Glucose Dulbecco's Modified Eagle Medium (DMEM) | ThermoFisher (Waltham, MA USA) | 13-1331-82 | Phenol red-free minimal essential medium Stock Concentration: 1'000 mg/l glucose |
Fetal Bovine Serum (FBS) | ThermoFisher (Waltham, MA USA) | 10500 | Stock Concentration:0.2 mg/mL Final Concentration: 2 μg/mL |
Prolong Gold antifade reagent with 4’,6-diamidino-2-phenylindole | Invitrogen (Waltham, MA, USA) | P-36931 | Aqueous mounting medium + DAPI Final Concentration: 1X |
Paraformaldehyde | Sigma (St. Louis, MO, USA) | P6148 | Fixative Final Concentration: 4% |
LAB-TEK two-well chamber slides | Sigma (St. Louis, MO, USA) | C6682 | |
Anti-Nestin antibody [clone 10C2] | Abcam (Cambridge, UK) | ab2035 | Stock Concentration: 1 mg/ml Final Concentration: 7 μg/ml |
Alexa Fluor 555 Phalloidin | ThermoFisher (Waltham, MA USA) | A34055 | Stock Concentration: 200 UI/ml Final Concentration: 5 UI/ml |
Triton X-100 | Euroclone (Milan, Italy) | EMR237500 | Final Concentration: '0,05% |
MesenPRO RS Medium (MSC-RS medium) | ThermoFisher (Waltham, MA USA) | 12746-012 | |
Alexa Fluor 488 anti-mouse SFX kit | ThermoFisher (Waltham, MA USA) | A31619 | Goat anti-mouse secondary antibody + Signal enhancer Stock Concentration: 2 mg/ml Final Concentration: 2 μg/ml |
Pasteur Pipette | Kartell Labware (Noviglio (MI), ITALY ) | 329 | |
StemMACS AdipoDiff Media | MiltenyiBiotec (BergischGladbach, Germany) | 130-091-679 | |
StemMACS OsteoDiff Media | MiltenyiBiotec (BergischGladbach, Germany) | 130-091-678 | |
Osteoimage Bone mineralization Assay | LONZA (Walkersville MD-USA) | PA-1503 | Hydroxyapatite specific fluorescent staining solution |
50mL Polystyrene conical tube | Greiner bio-one (Kremsmünster Austria) |
227261 | |
Nile Red | ThermoFisher (Waltham, MA USA) | N1142 | Fluorescent staining solution for lipids Stock Concentration: 100 mM Final Concentration: 200 Nm |
Glycerin | Sigma (St. Louis, MO, USA) | G2289 | Final Concentration: '50% |
Polistirene Petri dishes | Sigma (St. Louis, MO, USA) | P5606 | |
24-well plates TC-treated | Greiner Bio-one GmbH (Frickenhausen, Germany) | 662160 | |
Endothelial Growth Medium, EGM-2 BulletKit (EGM-2) | LONZA (Walkersville MD-USA) | CC-3162 | VEGF-rich endothelial cell growth medium |
Leica Qwin Image Analisys Software | Leica (Wetzlar, Germany) | Image analysis software |