Abstract
Human mesenchymal stem cells (hMSCs) are currently available for a range of applications and benefits and have become a good material for regenerative medicine, tissue engineering, and disease therapy. Before ex vivo expansion, isolation and characterization of primary hMSCs from peripheral tissues are key steps for obtaining adequate materials for clinical application. The proportion of peripheral stem cells is very low in surrounding tissues and organs; thus the recovery ratio will be a limiting factor. In this review, we summarized current common methods used to isolate peripheral stem cells, as well as the new insights revealed to improve the quantity of stem cells and their stemness. These strategies offer alternative ways to acquire hMSCs in a convenient and/or effective manner, which is important for clinical treatments. Improved isolation and mass amplification of the hMSCs while ensuring their stemness and quantity will be an important step for clinical use. Enlarged suitable hMSCs are more clinically applicable for therapeutic transplants and may help people live longer and better.
Keywords
Introduction
Human mesenchymal stem cells (hMSCs) are derived from embryonic mesodermal progenitors and can be isolated from tissues such as bone marrow (BM), umbilical cord blood, adipose tissue, muscle, corneal stroma, and tooth bud (14,55,74). These multipotent cells possess the self-renew capacity and are able to differentiate into many tissue types including osteoblasts, chondrocytes, adipocytes, neurons, and hepatocyte-like cells (65,77). Compared to human embryonic stem (hES) cell lines, autologously isolated hMSCs have the advantage of lower immunorejection, as well as a lower risk of pathogen transmission and tumorigenesis (70,91). Besides, mass production of hMSCs for clinical purposes can be easily achieved, as their self-renew and multipotent differentiation capacity can be maintained after many passages of culture (52,59).
Expanding knowledge on salient features of hMSCs in regenerative and immunosuppressive properties is accumulating, which provides for the development of new therapeutic strategies for clinical applications (12,54,74). hMSCs have been widely used in the study of many medical conditions including hepatic, cardiovascular, neurological, orthopedic, pancreatic, and immunological diseases (13,25,45,70,77). Stem cell-based therapies mainly function as cell replacement to restore degenerative or defective organs (10,31). Combination therapies include treating with small molecular drugs, for example, valproic acid (VPA), which stimulate proliferation and self-renewal of stem cells (7,22,68). For targeted delivery systems, hMSCs are considered an ideal vehicle to carry and effectively release therapeutic agents toward various malignant tissues (1,32,51,75). In neurodegenerative diseases or nerve injury issues, especially in Parkinson's disease, amyotrophic lateral sclerosis, stroke, and Alzheimer's disease, stem cell-mediated transfer has been widely used for gene therapy (4,26,43,50).
For MSC-based regenerative medicine, a well-established procedure for isolation, manipulation, and expansion of these cells is the key to success (5,93). hMSCs account for only 0.01% to 0.001% of nucleated cells; thus, effective isolation technologies are an important step toward clinical application (58,64). Inadequate quantities and poor stemness qualities of hMSCs would be limiting factors in the use of these cells for clinical therapy; therefore, improved isolation of the stem cells is desired (26,96). Suitable in vitro isolation conditions that resolve the problems of limited cell numbers and maintenance of stemness are necessary for clinical applications. In this review, we focused on issues that have been considered when manipulating hMSC techniques by varying isolation procedures. Further, we summarized improved methods and new insights that may help people to improve hMSC isolation and provide more materials for transplant medicine.
Establishment of HMSCS
hMSCs can be easily isolated from peripheral organs obtained in a relatively noninvasive manner, and their multipotentiality can be maintained for several passages (14,63,79). It was worth noting that the efficiency and quality of isolated hMSCs have variable effects in different mediums, procedures, temperatures, and oxygen tension (11,97). The simple method has been established for different kinds of hMSCs, which is mainly relayed on their adherence properties to achieve isolation (19,41). After plating of multipotent stem cells extracted from umbilical cord and BM, most of the non-hMSCs could be separated through continuous culture (72). For tissue types such as adipose and liver, hMSC isolation included an additional digestion step with collagenase (34,48).
Immunophenotypic analysis is necessary for the identification of the previously mentioned adherent cells and to allow for the further separation of nonmultipotent or nonconformance cells through their specific surface markers (53,67). It would be of utmost importance to identify the hMSC lineage, and this can be easily verified through flow cytometry (58,86). Although there is little consensus on the definitive markers of all types of hMSCs, data suggest that they still have some common cell surface markers. A minimal phenotypic pattern requires expression of cluster of differentiation 73 (CD73), CD90, and CD105, but not CD34, CD45, human leukocyte antigen (HLA)-DR, and other molecules (39,58,67). For specific immunophenotypic patterns, the variety of tissue sources of peripheral stem cells that could be isolated by their lineage-specific surface markers are summarized in Table 1 (59,98).
Surface Marker Expression Profiles of Main MSC Types [Updated From Review Article of Orbay et al. (59)]
Abbreviations: MSCs: mesenchymal stem cells; CD: cluster of differentiation; ADSCs: adipose-derived stem cells; BMMSCs: bone marrow-derived mesenchymal stem cells; HLA: human leukocyte antigen; PDLSCs: periodontal ligament-derived stem cells; TBMSCs: trabecular bone-derived mesenchymal stem cells; SMMSCs: synovial membrane-derived mesenchymal stem cells; PMSCs: periosteum-derived mesen-chymal stem cells; MMSCs: muscle-derived mesenchymal stem cells and satellite cells; Sca1: stem cell antigen 1; VCAM: vascular cell adhesion molecule; ICAM: intercellular adhesion molecule; SSCs: skin stem cells; SH2: CD105 antibody; SH4: CD73 antibody; EGFR: epidermal growth factor receptor; PDGFRa: platelet-derived growth factor receptor a; WJ-MSCs: Wharton's jelly mesenchymal stem cells; SH3: CD73 antibody; EpCAM: epithelial cell adhesion molecule.
Improved Isolation
The protocols for MSC isolation from various tissues are already well established; however, investigators are facing challenges in that the applicability of the primary MSCs obtained is relatively low (39,49). A prerequisite for ex vivo expansion is determining how to obtain a pure population of primary hMSCs rapidly and the identification of an optimized method for their isolation and purification (37). Current improvements include a lower segregation ratio of hMSCs and shorter times necessary to maintain stem cell properties during subculture of the cells (94). The methods are being continuously developed to obtain sufficient qualities, such as a unified cell type and multipotency (34,55). The current improvements in isolation methods have showed many new insights in acquiring better primary hMSCs for subsequent medical applications (23,55,88).
Vascular Perfusion for Liver Progenitors
Traditional isolation techniques to isolate MSCs from peripheral organs and tissues yield only a few available hMSCs and can cause cell injuries from collagenase usage. Reducing collagenase exposure time to prevent cells from undergoing enzymatic damage is therefore an important aspect in improving the quality of hMSCs (27,62). Experimental fetal liver donated from therapeutic abortions, an alternative resource to adult liver, has been used for isolating mesenchymal progenitor cells (44,69,84). Further, an improved method proposed that the vascular perfusion technique reduces the exposure of the tissue to collagenase (24). The method is mainly based on the procurement for perfusion via the portal vein with the adaptation of a five-step portal vein in situ perfusion method (24). They designed suitable solutions A to D and optimized other conditions, to separate mesenchymal progenitor cells from liver, and cultured hepatocytes in Williams' E medium-based Heparmed Vito 143 (Biochrom AG, Berlin, Germany) (24). In contrast to the static collagenase digestion, vascular perfusion obtained high viabilities of mesenchymal progenitors and greater cell numbers, as well as prolonged stemness (24,71).
Mechanical Dissociation of Lipoaspirate
Methods to isolate and culture adipose-derived stem cells (ADSCs) were developed extensively; however, little has been done to improve cell yields and multipotency (2,21,34). An alternative method, mechanical dissociation, has been used to isolate a population of hMSCs from lipoaspirate without collagenase treatments. The aim of the previous study was to increase yields and cryopreserve the cells before ex vivo expansion while maintaining multipotency for therapeutic application. Adipose tissue samples were incubated with ammonium– chloride–potassium (ACK) buffer solution and then shaken for preliminary red blood cell (RBC) lysis (2). The cells were subsequently cultured to select adherent cells, and ADSCs were isolated through flow cytometry analysis for the previously mentioned cell markers. They further observed that treated lipoaspirate samples could be stored without damage to the ADSCs during the mechanical dissociation procedure at 4°C (2). Mechanical dissociation therefore offered a convenient isolation method and allowed for large volumes of adipose separation, which are more suitable for commercial purposes (2,3,42,94).
Isolation of Adipose-Derived Cells From Blood/Saline Phase
Compared with the original isolation of ADSCs from adipose, which was frequently 8–10 h of continuous intense effort, another rapid separation method has been developed that occurs in less than 30 min (18,99). The basic principle relies on obtaining ADSCs from the blood/saline phase, which contains rich adipose-derived cells due to their perivascular origin rather than the oil phase of adipose extracts (9,76). A defined simple five-step process could isolate ADSCs from the more buoyant adipose tissue and show a mesenchymal morphology and immunophenotype (18). The method is both time saving and a simple technique for preparing ADSCs, which is critical for advancing transplant medicine therapeutics.
Novel Marrow Filter Device
A novel BM filter device has been explored to improve isolation methods, which collects nucleated cells without the use of continuous gradient centrifugation (61). Filtered BM is delivered into the device, which includes a carefully designed nonwoven fabric filter in a closed system. Most of the nucleated cells could be separated through a saline flow wash, removing RBCs and platelets (PLTs), and then harvested in defined collection medium (61). Such a closed workflow offered rapid purification duration times and also prevented a risk of contamination from external exposure during the operation. It also reduces operator input, favorably influencing the cell yields and qualities, and established a standard protocol for clinical cell therapy trials (28–30,60). This would represent a major advance in BM isolation by increasing the recovery ratio, thus allowing for less ex vivo expansion, from freshly harvested peripheral stem cell tissues (28,61).
Clot Spots Method
Wharton's jelly mesenchymal stem cells (WJ-MSCs) within human umbilical cord is a noncontroversial source of hematopoietic stem cells (HSCs), which could be used in transplant medicine (17,57). The properties of WJ-MSCs are comparable with those of fetal rather than adult- derived hMSCs and thus show more proliferative and immunosuppressive therapeutic effects (42). A new approach displayed an improvement in isolating WJ-MSCs and has since been recognized as a good source that could be suitable for clinical application (35). Different from the Rosset Sep method (35), the Clot Spot method used MesenCult™ complete medium (Stem Cell Technologies, Vancouver, BC, Canada) to culture primary human umbilical cord blood (HUCB) (46). Semisolid cord blood clots were explanted in the medium without disturbing the blood clots. The clots were subcultured for adherent cell selection and the cells identified morphologically and by immunophenotyping. In comparison to the Rosset Sep method, the Clot Spot method demonstrated a threefold increase of hMSC recovery from WJ-MSCs (35).
Stimulated BMMSCs Mobilized Into Peripheral Blood
Since bone marrow mesenchymal stem cells (BMMSCs), which are found in the interior of bones, are currently the most common source of hMSCs, their use is limited due to the painful harvest procedure and surgical risks (17,95). To prevent the need for invasive surgery, the in vivo mobilization of BMMSCs into the blood is an alternative method to harvest stem cells for transplantation (40). Fibrin microbeads (FMBs) could bind mononuclear cells and isolate hMSCs from human peripheral blood that had been mobilized from the bone marrow with granulocyte colony-stimulating factor (G-CSF). G-CSF alters the stem cell homing niches in the BM through reduced vascular cell adhesion molecule 1 (VCAM-1), stromal cell derived factor 1 (SDF-1), and stem cell factor (SCF) expression and causes marked downregulation of adhesion resulting in the release of MSCs from BM into the peripheral blood (47,90). This mobilization procedure efficiently results in MSC isolation and shows significantly lower contamination by other cell types (40,80). A combinational treatment of G-CSF with plerixafor, which reversibly blocks SDF-1 binding to chemokine C-X-C motif receptor 4 (CXCR4), improved the collection of MSCs compared with G-CSF alone (47,83).
Conclusion
In using hMSCs for regenerative medicine, core technologies must be established for preparing large numbers of suitable stem cells (78). Ultimately, if the key technologies were capable of providing an endless supply of stem cells, then stem cells could actually be applied in cell therapy (8,65). These technologies include acquiring a sustainable source from the peripheral tissues, the isolation of multipotent cells, and further ex vivo expansion (Fig. 1) (65). However, enhancing isolation methods may be a critical issue, due to inadequate sources of peripheral stem cells, so that a higher quality of hMSCs can be acquired (2,39). In summary, improved separation technologies need to have the following features: [1] prevent potential contamination during manipulation, [2] enhance the recovery ratio of hMSCs from limited source, [3] improve the time of stemness during passages, and [4] make the procedures more convenient and less costly, important parameters for a commercialized purpose. Taken together, refining isolation strategies of hMSCs can increase their clinical applicability by improving primary stem cell yield and so reduce the need for ex vivo expansion (Fig. 1).
The relationship of human mesenchymal stem cell (hMSC) separation technology with its potential clinical application. Isolation methods influence operating times and the hMSC quantities, both qualities that are important for transplant medicine.
Footnotes
Acknowledgments