| Introduction:The human mesenchymal stem cells (hMSCs) are non-hematopoietic stromal progenitor cells, capable of maintain their self-renewal and differentiating into some mature cells of multiple mesenchymal tissues including body fat, bone, cartilage and muscle. The hMSCs isolated from bone marrow are also called marrow stromal cells. In recent years, the multiple differentiation potentials of hMSCs which just like ES cells (Embryonic Stem Cell), coupled with the ease of in vitro culturing has spurred considerable interests in better understanding the biology of hMSCs and their potentially therapeutic and clinical research applications.In our research project we believe that the prostate stromal cells provide a niche and a microenvironment for occurrence, progression and treatment of benign prostatic hyperplasia (BPH) and prostate cancer (CaP). Meanwhile, they form a pool of precursors for endothelial, myogenic, adipocytes, chondrocytes or osteoblasts cells which may include the most primitive, mesenchymal stem cells (MSCs), or as proposed recently multipotential stromal cells. Despite these increasing interests, compared with human prostate epithelia stem cells (HPESCs), our understanding of hMSCs biology remains rudimentary. In fact, hMSCs maintained in different laboratories or hMSCs isolated from different species are various in their characteristics. There are many factors confounding the understanding of the characteristics of hPMSCs and their differentiation potentials. First, during the individual development, hPMSCs have different phenotypes and functions. Second, there are subpopulations of hPMSCs in bone marrow, differing by the stage of their differentiation. Third, hPMSCs at different sites in the bone marrow may be different. Fourth, different isolating methods and culture conditions may affect the characteristics of the hPMSCs maintained. Finally, the induction paradigm used in vitro and in vivo may affect their differentiation potentials. To date, there is no specific marker or combination of marker to identify hPMSCs either in vivo or in vitro, and there is no standardized procedure to isolate hPMSCs. Our experimental objects are to explore and develop the methods to isolate the human prostate mesenchymal stem cells (hPMSCs) from prostate stroma in benign prostatic hyperplasia (BPH) to identify their distinct biological features. Hopefully this may lead us to enrich the concept of prostate cancer stem cells, as well pave a new way for the diagnosis and therapy of clinical BPH. Aim:Under these consideration, we initiated our study by establishing clonal hPMSCs cell lines from these PA-hPMSCs (Plastic Adhesion-enriched hPMSCs),HPMNCS(Human prostate stromal mononuclear cells), CD105, CD133and CD271magnetic beads sorted cells, and uncovering culture conditions to keep hPMSCs at undifferentiation state. We had harvest the hPMSCs directly isolated from human prostate by using MACS (Magnetic activated cell sorting) then went to characterize these hPMSCs cell lines and identify clones for further investigation. Finally, we evaluated the multi potential differentiation capability of these hPMSCs into Adipo, Osteo and Neural lineage in vivo and in vitro. To demonstrated that immuno-magnetic enriched of cells expressing CD271(LNGFR) and CD133could be used for the purification of hPMSCs and for the establishment of highly proliferative and multipotential HPMSC cultures, they are the most specific markers for the prospective isolation of highly purified hPMSCs. Overall, we conclude that hPMSCs from human prostate may be an ideal stem cell source for cell therapies for prostate disease and tumor related clinical research.Materials and Methods:hPMSCs were isolated from the patients with BPH (Benign Prostatic Hyperplasia) undergoing Trans Urethral Resection of the Prostate (TURP). All the five postsurgical samples were prior consent obtained according to the procedures approved by the Ethics Committee of the University of Edinburgh.The prostate tissue samples of the patients with BPH (n=5) were enriched for hPMNCs by Percoll (d=1.073g/ml) gradient centrifugation and subsequently the cells (passage0) harvested from interphase was collected and cultured to passage1. Then the expended passage1cells was divided into three parts (3/5,1/5and1/5) and processed as following. Accordingly, the part containing3/5of hPMNCs passage1was divided into three equal parts. One equal part of these cells was directly subjected to isolation of CD105, CD133and CD271positive cells, respectively. Another1/5of hPMNCs passage1was employing Plastic Adhesion-enriched hPMSCs sorting protocol. The remaining hPMNCs passage1was cultured and expended to passage3directly.The data on all5hPMSCs frictions are obtained after prolonged culture in vitro. Thus, we also cultured PA-hPMSCs (Plastic Adhesion-enriched hPMSCs),HPMNCS, CD105, CD133and CD271magnetic beads positive sorted hPMSCs for three passages to perform further evaluation of each fraction. Then, all of five colony cell lines were established after expansion to passage3in vitro. These cell lines were systematically characterized and their differentiation capabilities were evaluated. All of the five cell lines that has the classical characteristics of hMSCs and was able to differentiate into lipoblast-like cells and osteoblast-like cells in vitro was further analyzed by evaluating their Oil Red O and Alizarin Red S staining ability. The methods are as followed:Counting the colonies of CFU-F:For assess the capability of HPMSC to form CFU-F colonies, all cells were cultured with MACS NH CFU-F Medium (#130-091-676) for9days in1%gelatin coated6wells plate. On day9the flasks were rinsed, fixed with methanol and stained with crystal violet staining solution and the number of colonies was counted (CFU-Fs represent stem and progenitor cells with non-hematopoietic differentiation potential.).Flow cytometry test:The phenotype of cells from4fractions was analyzed with the panel of mouse mAb specific for MSCs like CD73, CD44, CD90, the specific positive phenotype of cells for HPMSC as CD73, CD105and CD166and the markers for hematopoietic cells like CD14, CD34, and CD45. Briefly, to1×105cells suspended in100μl of staining buffer (D-PBS,1%BSA)20μl of mouse mAb was added. Next, the cells were incubated in the dark for30min at4℃. Stained cells were washed, collected using Coulter EPICS XL/XL-MCL Flow Cytometry System (Coulter Co, Florida, USA) and analyzed, was also be tested.To induce hMSCs into osteoblast differentiation, we have used1×105cultured cells were plated in6-well plate (Sarstedt Co, Newton USA) in Nonhematopoietic OsteoDifferentiation Medium (Miltenyi Biotec Co, Germany, order no.130-091-678). After9days induction, the shape of hMSCs changes from spindle-shape to cuboid-shape with smaller nuclear to cytoplasm ratio. Calcium-phosphate deposits could be observed. Cells were cultured for14days and refeeded twice per week. To demonstrate osteogenic differentiation, the cultures were washed with phosphate-buffered saline, fixed with4%paraphormaldehyde for2min, washed with distilled water, stained with alizaryn red S for1min. and finally washed with ethanol in order to assess calcium phosphate deposits, evaluating their Alizarin Red S staining ability by OD reading (MRX(?) Ⅱ ELISA (Dynex Co, USA)) respectively.To induce lipoblast differentiation,1×105cultured cells were plated in6-well plate (Sarstedt, Newton USA) in Nonhematopoietic Adipo Differentiation Medium (Miltenyi Biotec, Germany, order no.130-091-677). Cells were cultured for4weeks with twice weekly refeeds. The cells containing lipid vacuoles were observed after3weeks, evaluating their Oil Red O staining ability by OD reading (MRX(?) Ⅱ ELISA, Dynex Co, USA) respectively.Neurocytes differentiation. To induce neuro differentiation,1×105cultured CD271positive cells were equally plated in12-well plate in Nonhematopoietic N2B27Medium. NDiff(?) N2B27(?)(Stem Cell Sciences Co, USA, Catalogue Number:SCS-SF-NB-02).The cells was cultured in N2B27with β-NGF (Nerve Growth Factor-β,30ng/ml), FGF-b (Fibroblast Growth Factor-basic,20ng/ml) and RA (All trans-retinoic,0.5ug/ml) for72hours. Fixed specimens were analyzed with fluorescence microscopy, and expression of nestin, GFAP at48hours and72hours was evaluated by immuno-fluorescence and PCR (polymerase chain reaction). To demonstrate neural differentiation, the culture wells were washed with phosphate-buffered saline, fixed with4%formaldehyde for5mins, washed with distilled water. Firstly, the cells are fixed and then non-specific proteins are blocked. Next, two antibodies were added to the cells that have been produced in different species. Finally the cells were stained with fluorescently labeled secondary antibodies for30mins enabling the proteins to be visualized by fluorescent microscopy.Results:In this study we have evaluated five cell fractions:1) non-purified hPMNC (Human prostate stromal mononuclear cells),2) Plastic Adhesion Enriched cells (PA) in hPMNC isolated by their plastic adherence properties.3) CD105magnetic beads positive sorted hPMSCs.4) CD133magnetic beads positive sorted hPMSCs.5) CD271magnetic beads positive sorted hPMSCs for:(1) Growth characteristics of Cells,(2)their efficacy to form CFU-F colonies,(3) Expression of MSC-associated antigens (CD105, CD166, CD44, CD73, CD45, CD34) and mDNA (NESTIN, GFAP) for PCR test.(4) their neural/adipocyto/osteogenic potential in vitro. The results are as followed:MACS (Magnetic activated cell sorting) results:To determine the possibility and efficiency of purification of CD133, CD271and CD105positive stem cells from human prostate stromal by using immunomagnetic beads, the single cell suspension of human prostate tissue of the patients with BPH (Benign Prostatic Hyperplasia) undergoing Trans Urethral Resection of the Prostate (TURP) was prepared and incubated with PE labeled anti-CD133antibody. The labeled cells were subsequently incubated with anti-PE magnetic beads and separated through magnetic LS and MS cell sorting system. The rate of CD133positive cells in pre-and post-separation groups was assessed by flow cytometry. The results showed that the purity of CD105positive cells amounted to (97.3±2.8)%after separation, CD133positive cells amounted to (88.3+3.06)%after separation, CD271positive cells amounted to (89.5±1.8)%after separation. The viability of cells was not significantly impaired (P>0.05) during the. MACS (Magnetic activated cell sorting) purification process. The study indicates that the highly purified and viable CD105, CD133and CD271positive stem cells can be directly isolated from human prostate by using MACS (Magnetic activated cell sorting) in combination with cd105, CD133and CD271superficial marker.All of5isolated colony cell lines have basic features of human tissue mesenchymal stem cells (hMSCs). Under phase-contrast microscope, these hPMSCs cells were homogeneously small spindle-shaped and rare little broader cells were seen among them. We used10%FBS in-MEM medium to culture hPMSCs, and transfer of culture after it reaches80%confluence. Growth curve was drew at passages3and the population doubling (PD) time was26+2.3h by calculation.CFU-F result:The five groups of isolated cells were seeded into6wells flacks and cultured in MACS(?) NH CFU-F Medium (Miltenyi Biotec Co, Germany, order no130-091-676)for9days. We noticed that the CD133+and CD271+cells isolated by immunomagnetic beads yielded the highest number of CFU-F colonies which was51±7.31and76±10respectively. Accordingly, the number of CFU-F colonies from CD133+and CD271+fractions was3-4times higher as compared to the other isolated hPMSCs groups. Interestingly, CFU-F formed by purified CD271and CD105fraction proliferated most rapidly and the cultures initiated by these cells became confluent faster in comparison to other isolated hPMSCs groups.FCM (Flow Cytometry) results:hPMSCs expressed various markers for different lineage cells including mesenchymal cells, but not hemopoietic stem cells. By using flow cytometry, we found that these cells expressed the following set of gene markers (cell surface antigens):CD44, CD73, CD90,CD105,CD166, but not CD14, and CD45. These results showed that hMSCs do not express markers of hemopoietic stem cells (CD34, CD45) and CD14, either, but do express markers of mesenchymal stem cells (CD44, CD73, CD90) and CD105.All the five cell fractions were plated to grow and expanded to three passages, all which were subsequently plated in the cultures promoting osteogenic and adipogenic differentiation and after14days and28days stained with Alizarin S and Oil Red to visualize calcium-phosphate deposits and presence of lipid droplets, respectively. As similar to their CFU-F ability, the OD reading (MRX(?) Ⅱ ELISA, Dynex Co, USA) from CD133+and CD271+fractions was also2-3times higher as compared to the other isolated hPMSCs groups.In vitro differentiation study showed that hMSCs can differentiate into neural-like cells, lipoblast-like cells and osteoblast-like cells. To induce CD271+hPMSCs into neural differentiation, we use cultured in N2B27Neuro-Diff medium withβ-NGF (Nerve Growth Factor-β,30ng/ml), FGF-b (Fibroblast Growth Factor-basic,20ng/ml) and RA (All trans-retinoic,0.5ug/ml). After36hours of induction, fibroblast-like hPMSCs started to exhibit neuron-like morphology, extending long processes terminating in typical growth cones and filopodia. Further immunofluorescence analysis and PCR express revealed that the differentiated cells express following neuron makers: Nestin, and GFAP (Glial Fibrillary Acidic Protein). By using these N2B27Neuro-Diff medium with β-NGF, FGF-b and RA we can maintain the viability of the cells above94%after induction more than14days.The PCR results showed that the expression of CD271and Nestin was positive in neuron-differentiated CD271positive cells. The neuron-diff CD271positive cells displayed also the expression of GFAP. Furthermore, the Nestin and GFAP were never expressed in non neuron-diff control group.In conclusion, all the5clonal cell lines of hPMSCs can differentiate into neuro-like cells. lipoblast-like cells and osteoblast-like cells in vitro.Several tests of evidence suggest these hPMSCs cells are hMSCs. First, even after in vitro culture over35PD, these cells have the capacity for self-renewal. Second, these cells expressed various markers for different mesenchymal lineages, but not the marker from hemopoietic stem cells. In addition, they maintain the ability to give rise to three different types of differentiated mesenchymal lineage progeny (osteogenic cells, adipogenic cells and neurogenic cells). Therefore, we will call them hMSCs there after.Statistical Analysis:Data for number of cells seeded, number of CFU-F colonies, expression level of chosen antigens are represented by the mean+SEM (χ±s). The difference between means was performed with Student’s T test. All statistical analyses were performed using SPSS11.0software(Chicago, IL, USA). p<0.05was considered as statistically significant.Conclusion and Discussion:To our knowledge, this is the first time we establishing a simple sequential procedures to isolate multi-potential HPMSC lines from human prostate tissue of the patients with BPH (Benign Prostatic Hyperplasia) undergoing Trans Urethral Resection of the Prostate (TURP). We have also, for the first time, demonstrated that hPMSCs from adult human prostate by simple sequential procedures of isolation and culture system have the ability to differentiate into neuro-like cells, lipoblast-like cells and osteoblast-like cells in vitro. We also discovered that the same hPMSCs from different sorting protocol assume different fate under Molecular Bio-analysis in vitro. It is worth noting that even after long term in vitro culturing all five hPMSCs frictions still maintain the capacity for self-renewal and multi-potential differentiation capability. These characters allow sufficient time for further in vitro manipulation of these stem cells before further clinical applications and further research. And these cytological characters of hPMSCs are much similar like the hMSCs from bone marrow and plancenta."Friedenste AJ,1970and Gronthos S,2003observed that single-bone marrow stromal cell adherent to plastic culture dish can form colony of cells morphologically resembling fibroblasts in vitro, called clonogenic progenitor cells or Colony forming units-Fibroblast (CFU-F). They further demonstrated that the in vitro growth of different adherent colonies are heterogeneous and only cells from some colonies may be multi-potential hMSCs. However, like all the other current isolation procedure, this method of isolation yields a heterogeneous cell population. These plastic adherent cells also known as PA-hPMSCs (Plastic Adhesion-enriched hPMSCs), exhibit widely varying growth kinetics, cell markers and differentiation capabilities. We will use PA-hPMSCs as a target to compare with the cells harvest from the other) isolation procedures.In this study we compared side by side expansion and differentiation efficacy of hPMSCs isolated by employing three different commercially available isolation strategies to Plastic Adhesion-enriched based and magnetic activated cell sorting protocol. Our data reveal that hPMSCs purification using various isolation strategies results in different proliferation capacity of these cells and different patterns of differentiation efficacy. Positive selection strategies based on use of CD133and CD271magnetic antibodies yielded3-4times more CFU-F colonies respectively in comparison to hPMNCs and population enriched for HPMSC by Plastic Adhesion-enriched selection and CD105magnetic antibody activated cell sorting. Consequently, cultures started from CD133+and CD271+cells reached confluence and proliferated faster than those initiated isolated hPMSCs fractions. The possible explanation for this kind of difference is the fact that when cultured without accompanying hematopoietic cells present in purified CD271and CD105fraction fractions, proliferation potential of CD133and CD271cells is unlashed. This suggests that the growth of these cells is inhibited by haematopoietic cells present in other fractions. It is also possible that lower seeding density is advantageous in HPMSC expansion, for which they will yield lowest contact suppression. In the light of the culture we observed that CD271and CD133expression declined between initial populations of purified CD271and CD133friction to passage3. The possible reason to this might caused by to saving their stem-like ability by decrease rapidly during culture.Phenotype evaluation revealed that CD271+and CD133+populations contained the lowest hematopoietic contamination as judged by presence of CD45positive cells, which were1.66%and2.17%. Also CD105fraction has on average lower number of CD45+cells (4.39%)in comparison to HPMNC and PA-hPMSCs populations. Lack of hematopoietic contamination persisting until the third passage might be at least in part responsible for observed faster CD271+and133+populations’ high proliferation and differential abilityThe ideal marker for hPMSCs should exhibit a high level of expression on rare MSCs and minimal expression on cells of the predominant hematopoietic lineages (HLCs). Our study demonstrated that immuno-magnetic enriched of cells expressing CD271(LNGFR) could be used for the purification of hPMSCs and for the establishment of highly proliferative and multipotential HPMSC cultures. But the microbeads sorting results are not as good as expected, the highest percentage we ever made (MASC CD271microbeads kit-APC) is about20.1%positive by run instant FACS analysis (use the freshly sorted positive cells to run Flowcytometry Analysis.).As the new frontier of HPMSC research, the standardization of isolation, expansion, and differentiation protocols are needed. Our study shows that with respect to HPMSC isolation from humans post operation BPH samples, CD271(LNGFR) immuno-magneticbeads based methods hold great promise. Our data clearly displayed the potential of CD271(LNGFR, Low affinity nerve growth factor receptor) as a novel marker of proliferative and multipotential hPMSCs from human prostate stromal. In addition, these methods can also be used for the isolation of hPMSCs in prostate cancer (CaP). Our study demonstrated that immuno-magnetic enriched of cells expressing CD271(LNGFR) and CD133could be used for the purification of hPMSCs and for the establishment of highly proliferative and multipotential HPMSC cultures. Overall, we conclude that hPMSCs from human prostate may be an ideal stem cell source for cell therapies for prostate disease and tumor related clinical research. |
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