عزل خلايا Mesangiogenic السلف (البلدان المتوسطية الشريكة) من نخاع العظام البشرية
Summary
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.
Abstract
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”.
Introduction
خلايا انسجة الوسيطة (اللجان الدائمة) هي القيمة السريرية ذات الصلة لقدرة التمايز متعددة نسبهم وقدرتها على دعم تكون الدم، لإفراز عوامل النمو / السيتوكينات وكذلك تلعب دورا في يممونوريغولاتيون 1. في تعريف إنتاج خلايا العلاجات القائمة MSC-والطلب قد يكون هدفا للالسريرية وما قبل السريرية بحث مستفيض 2، مع إيلاء اهتمام خاص لتنظيم دولي محدد من أجل سلامة وفعالية الخلايا المنتجات الطبية (CBMP) العلاجات 3 شهادة. اللجان الدائمة الإنسان يتم تربيتها على نطاق واسع في وسائل الإعلام التي تحتوي على ملاحق والكواشف ذات الأصل الحيواني، مثل مصل الجنين البقري (FBS) والتربسين البقري. لذلك، جنبا إلى جنب مع مع المخاطر المعدية المرتبطة معالجة الخلية، والمرضى أيضا تواجه بريون التعرض فضلا عن المخاطر المناعية المرتبطة البروتينات والببتيدات أو غيرها من الجزيئات الحيوية من أصل حيواني يمكن أن تستمر بعد حصاد الخلايا وtransplanالكساء 4.
للالتفاف على المشكلة، ونحن مثقف نخاع عظام بشرية (HBM) اللجان الدائمة -derived في المتوسط خالية من الحيوانات، لتحل محل FBS مع المجمعة البشرية نوع AB مصل (PhABS). في ظل هذه الظروف، إلى جانب اللجان الدائمة المتزايد حددنا سكان الخلية الرواية. وكانت هذه الخلايا شكليا وظاهريا مختلفة من اللجان الدائمة وأظهرت لمحة التعبير الجيني المميز وكذلك المميزة خصائص النمو / الالتصاق. احتفظوا كلا mesengenic وعائية إمكانية وبالتالي سميت خلايا أرومية متوسطة السلف (البلدان المتوسطية الشريكة) 5. وفي وقت لاحق، كنا قادرين على تحديد شروط ثقافة انتقائية وقابلة للتكرار لتوليد البلدان المتوسطية الشريكة في درجة عالية من النقاء 6.
ونحن التحقيق كذلك الخصائص المورفولوجية والبيولوجية من البلدان المتوسطية الشريكة. أظهرت البلدان المتوسطية الشريكة أن تكون nestin إيجابية، وبطء في ركوب الدراجات، كي-67-سلبي، ومع الكروموسومات التي تتميز التيلومير طويلة 5. وأعربوا عن plurعوامل النسخ المرتبطة ipotency-أكتوبر-4 و NANOG بدلا من لجنة السلامة البحرية المنظمين سيد Runx2 وSox9 7. ظاهريا، أعرب البلدان المتوسطية الشريكة endoglin (CD105) في مستوى أدنى من اللجان الدائمة في حين تفتقر علامات الوسيطة CD73، CD90، CD166. كما أظهرت البلدان المتوسطية الشريكة نمط مميز من جزيئات الالتصاق تتميز التعبير ثابت من PECAM (CD31)، integrins αL (CD11a)، αM (CD11b)، αX (CD11c و) وكذلك إنتغرين β2 (CD18) أن يديم على وجه التحديد تشبه الهياكل podosome 8 . في وسائل الاعلام التوسع MSC القياسية، البلدان المتوسطية الشريكة متباينة على وجه السرعة إلى اللجان الدائمة من خلال مرحلة وسيطة يضم تفعيل خلية Wnt5 / كالمودولين يشير 9. كما احتفظ البلدان المتوسطية الشريكة خصائص عائية، كما يتبين من قدرتها على الانطلاق من الكروية في الفئران المصفوفة خارج الخلية (ECM) الثقافات البروتين 3D. وكان احتمال عائية فقدت بسرعة بعد التمايز لجنة السياسة النقدية على طول النسب mesengenic.
هنا نقدم محترفينtocols الأمثل لعزل وتوصيف البلدان المتوسطية الشريكة العالية النقاء من عينات الدم HBM. ويرد أيضا بروتوكولات استنساخه لجنة السياسة النقدية mesengenic والتمايز عائية.
Protocol
Representative Results
Discussion
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.
Disclosures
The authors have nothing to disclose.
Acknowledgements
فإن الكتاب أود خصوصا أن أشكر الدكتور باولو Parchi، قسم الجراحة والطبية وعلم الأمراض الجزيئية والعناية المركزة، جامعة بيزا، لتوفير عينات نخاع العظام وخبرته في أستيو-الأسلاف البشرية
Materials
| 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 |
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