Isolation and characterisation of Mesenchymal Stem Cells from Human Peripheral Blood

Aim: We describe a comprehensible novel procedure for isolation and characterization of mesenchymal stem cells (MSCs) from human peripheral blood. Background: MSCs are well known multipotent adult stem cells with a wide spectrum of divisibility including osteoblasts, chondrocytes, myocytes, adipocytes many isolations and characterization techniques have emerged to isolate MSCs from human peripheral blood, most which are expensive and time-consuming. Methods: MSC isolation was carried out from human peripheral blood and cultured in a well-defi ned culture medium. For adherence of culture, cultures were maintained at 37 0C in a humidifi ed atmosphere containing 5% CO2 for 48h. Thereafter, medium was replaced with fresh one every 3-4 days for 21 days. Adherent cells were characterized by fl ow cytometry verifi ed MSC specifi c markers. Results: Isolated MSCs had a fi broblastic-like appearance with adherent property to the culture plate. After isolation of MSCs, purifi ed MSCs from peripheral blood were seen to be positive for positive cell surface markers: CD73, CD90, and CD105 while being negative for CD14, CD34 and CD45 along with their respective conjugates. Conclusion: These fi ndings affi rms that the above method described is capable of isolating MSCs from human peripheral blood with positive results of stem cell characterization through fl ow cytometry according to all minimum criteria defi ned by the International Society for Cellular Therapy (ISCT). Research Article Isolation and characterisation of Mesenchymal Stem Cells from Human Peripheral Blood Subhajit Hazra1,2§*, Aleya Tabasum1, Manasseh Nithyananth3 and Murugan Ramalingam1,4 1Centre for stem cell research (CSCR), A unit of Institute of Stem Cell Biology and Regenerative Medicine-Bengaluru, Christian Medical College Campus, Vellore 632002, India 2Department of Pharmacology, Gupta College of Technological Sciences; Asansol 713301, India 3Christian Medical College (CMC), Vellore, Tamil Nadu, India 4WPI-Advanced Institute for Materials Research, Tohoku University, Sendai 980-8577, Japan §Former Short-Term Trainee at CSCR; *Technical Staff at cGMP (CSCR) Received: 18 June, 2018 Accepted: 14 July, 2018 Published: 16 July, 2018 *Corresponding authors: Subhajit Hazra, Centre for stem cell research (CSCR), A unit of Institute of Stem Cell Biology and Regenerative Medicine-Bengaluru, Christian Medical College Campus, Vellore 632002, India, E-mail:


Introduction
Stem Cells (SCs) are defi ned as a "class of undifferentiated cells that has the ability to differentiate into specialized cell types each with their new characteristically specialized cell functions [1]. MSCs are multipotent adult SCs that are present in multiple tissues; including umbilical cord, bone marrow and fat tissue. MSCs can divide and differentiate into multiple tissues including bone, cartilage, muscle and fat cells, and connective tissue [2]. They have been derived from varied sources as bone marrow [3], human peripheral blood (hPBSCs) [4], adipose tissue [5], lungs [6], heart [7], placenta [8], cord blood [9], umbilical cord [10] and urine [11]. Despite of their easy culturing techniques, the process of isolation of MSCs from patients is complicated, painful [12], thus making it hard to fi nd donors. Isolation of SCs from human peripheral blood can thus provide a less complicated and painful alternative for stem cell therapies involving autografts and allografts. In case of allografts involving SCs, hPBSCs allografts have been seen to contain 3-4fold greater CD34+ cells as well as a 10fold elevation in number of lymphoid subsets count when mobilized with recombinant human granulocyte colonystimulating factor (rhG-CSF). Besides, it has been seen that peripheral blood stem cell transplant (which require hPBSCs) gives a better disease-free survival rate to patients suffering from advanced stage cancer than that of bone marrow derived MSCs [13]. Hence, for clinical applications; the hPBSCs seem to be more promising only if a solution to effectively isolation and culture of peripheral derived MSCs in vitro can be found. Thus, this research is focused on developing a novel and effective procedure or the isolation of hPBSCs from human peripheral blood.

Isolation of human mesenchymal stem cell from human peripheral blood
The study was conducted after obtaining approval from the Institutional Review Board (IRB) of Christian Medical College; CMC, Vellore. Male participants (volunteers), between 18 and 30 years were screened, and medical history and examination, electrocardiograph, hematology, biochemistry, serology, urine analysis, and chest X-ray were done within 14 days before start of the study. Participants having a history of major illnesses, acute or chronic disease, habituated to tobacco or alcohol were excluded from the study. After being given detailed information about the study, the participants were requested to sign an informed consent form after affi rming them that there was no harm was assigned to them. Human peripheral blood sample was thus obtained from donors as per procedure suggested by Jean M. Slockbower [14]. Venous blood was collected through venepuncture from volunteers into sodium-heparin tubes fi gure 1. Briefl y, at fi rst a suitable site of venepuncture was selected, tourniquet was placed 3 to 4 inches above the selected puncture site. Caution was maintained not put the tourniquet on too tightly or leave it on the donor for longer than a minute. Thereafter vein was selected, cleansed with 70% isopropyl alcohol in a circular motion. Subsequently, donor's arm was grasped fi rmly using thumb to draw the skin taut and anchor the vein. Needle was inserted swiftly through the skin into the lumen of the vein. Care was taken to see that the needle formed a 15-30-degree angle with the arm surface. Excess probing was avoided. After the tube got fi lled, needle from the donor's arm was withdrawn using a swift backward motion. Next, puncture site was gauzed. Thereafter, adequate pressure was applied to avoid formation of a hematoma. After holding pressure for 1-2 minutes, a fresh piece of gauze was taped to the puncture site. The blood so collected was uniformly mixed with heparin by a gentle tilt.
Mononuclear cells (MNCs) were isolated from peripheral blood by procedure as described hereafter. Briefl y, 5ml of blood was taken in a 15 ml centrifuge tube. To it 5ml of phosphate buffered saline (PBS) was added. To fi coll-paque, PBS-blood suspension was added in a ratio of 1:2. Thereafter, fi rst round of centrifugation (Figure 2), was done at 400Xg at room temperature for 30 minutes. Subsequently plasma layer was discarded and MNC layer collected. Caution was maintained in collection of MNC layer to avoid mixing of MNC layer with fi coll or plasma layer. Following collection of MNC layer a second round of centrifuge with MNC layer alone was performed at 700Xg for 10 minutes. After 2 nd round of centrifugation cell pellet was collected and to it 10% complete FBS media was added. The pelleted MNC's were cultured for 21 days. It is to be noted here that cultures were maintained at 37  C in a humidifi ed atmosphere containing 5% CO 2 . Suspended cells were discarded after 5-7 days of culture. Adherent cells were left to grow on the fl ask surface. Culture medium was changed every 3 days till 21 days.

Characterization of human mesenchymal stem cell through fl owcytometry
MSCs express CD105, CD73, and CD90 and are void of CD45, CD34, CD14 expression [16]. Flow cytometry was used to characterize hPBSCs using stem cell surface marker antibodies fi gures 5,6. Figure 5 shows the grouping of around 6*10 3 cells from around a cell population of 1*10 4 cells for fl ow cytometry analysis. Figure 6 shows results of fl ow cytometric analysis for the typical surface markers expressed in hPBSCs. Events P3, P5 and P7 signals from hBMSCs surface marker antibodies and events P2, P4 and P6 represent the isotype controls for fl uorescence from that negative markers and background.
Had the cell not been SCs one would not fi nd signals from hBMSCs surface marker antibodies, thus confi rming that the cells isolated from human bone marrow belonged to human mesenchymal stem cell lineage [17].

Conclusion
Mesenchymal stem cells are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells which give rise to marrow adipose tissue). To the best of our knowledge the novel procedure being investigated, is the fi rst of its kind for the isolation of MSCs from human peripheral blood. This endeavour was based in the backdrop of current scenario whereby it was suggested that there are few clinically relevant techniques for hPBSCs isolation and that hPBSCs is better than bone marrow derived MSCs in many ways. hPBSCs were isolated from blood of donors, characterized morphology analysis using inverted microscope and the results of the same