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LETTER TO THE EDITOR |
a Department of Biological Sciences,
b Division of Bioengineering, and
c Department of Orthopedic Surgery, National University of Singapore, Singapore, Republic of Singapore
Key Words. Bone marrow • Differentiation • Sieve-separated • Purification • Isolation
Correspondence: Dietmar Werner Hutmacher, Ph.D., M.B.A., Division of Bioengineering, Faculty of Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore. Telephone: 65-6874-1036; Fax: 65-6872-3069; e-mail: biedwh{at}nus.edu.sg
The ability and efficiency of various techniques to purify adult stem cells from a heterogeneous cell population is an important determinant for the successful characterization and application of stem cells. Strategies employed to isolate or purify such cells are numerous, and these include sorting of cells according to specific cell surface markers using either fluorescence [1] (the current gold standard in purifying stem cells) or immunomagnetic technology [2], exploiting the differential plating efficiencies of stem cells on culture plastic [3], and column-separation techniques [4]. Adding to this, a strategy reported by Hung et al. [5] uses "double-decker culture plates" with "3-µm" pores to separate stem cells from a heterogeneous population.
Hung et al. described plating Percoll gradient separated bone marrow stem cells on double-decker culture plates (Transwell, Corning, NY) where the top plate was fabricated with 3-µm pores. Size-sieved (SS) postcultured cells on the upper plate were described to be fibroblastic in morphology and large in size, while the lowere plate (LP) cells were small and polygonal. A series of assays characterizing the differentiation potential, proliferation, and a selected group of surface markers was reported, but only on the SS cells. Hung et al. suggested that the 3-µm pores separated the stem cells from the heterogeneous cell population. The true advantage and attractiveness of the proposed strategy would be its cost effectiveness, straightforward and user-friendly protocol.
However, it would be more convincing to the scientific communities of stem cell and tissue engineering if the LP cells were also characterized and shown not to possess the same differentiation potential and characteristics. This would be important before one can arrive at the conclusion that successful "sieving" of stem cells had occurred. The "stem-ness" of the separated cell population subsets (SS, LP) would directly correlate with the efficacy of the proposed strategy. The greater the efficacy, the more localized stemness would be on SS cells and the less on LP cells. Consequently, this would mean that the SS to LP data justify and reflect the efficacy of the procedure. Reporting SS cells as having stem cell characteristics may not be adequate because without sieve separation, the multipotency of bone marrow–derived stem cells have already been reported [6–10], thus leading us to expect some degree of stem cell characteristics with the SS cells anyway. Confirming the stemness of a stem cell population must precede its characterization. Without such confirmation, any conclusions arising from the characteristics of the purported stem cell population would be incomplete, and the derived clinical applications would have profound repercussion throughout the field. Ideally, research should assess stemness in terms of cellular self-renewal ability–the differentiation of in vitro and in vivo single-cell clones into cell types of the tissue of origin and at least one other different tissue cell type [11].
In addition, the characterization of LP cells would also provide some insights to stem cell biology. The presupposition of the proposed strategy is that stem cells in suspension would be larger than 3 µm and would thus remain in the upper plate (SS cells). Nevertheless, there are reports of stem cells having small diameters [12,13]. A number of other groups claim that "spore-like" [12] cells, found in virtually every tissue in the body, are of less than 5 µm diameter. These cells exhibited differentiation potentials. Young et al. [13] isolated clones of cells of very small diameters, derived from rat skeletal muscle. They reported that these clones had characteristics that are similar to those of embryonic stem cells, with a high nucleus to cytoplasm ratio and with molecular and immunological markers that are related to embryonic stem cells, and that they have high telomerase activity and extended doubling capacities. Based on this background, it might be argued that these "small" stem cells might reside in bone marrow, too, and would have filtered through the 3-µm pores, thereby emphasizing the value and need of characterizing the LP cells.
Additionally, to further validate the success of the sieve strategy in separating stem cells from other non–stem cells in the population, one might also investigate the presence and characteristics of non–stem cells, in particular, in the SS fraction. It is challenging to comprehend this method as a purification strategy for marrow stem cells as other cells found in the marrow are all too large for the "3-µm" pores; examples include lymphocytes (9–15 µm), granulocytes (12–15 µm), nucleated red cells (6–12 µm), and monocytes (16–20 µm) [14]. Based on the pore size of 3 µm of the upper plate, these contaminating cells would still be retained in the SS fraction but would be unseparated from the stem cell population. Furthermore, based on Table 1 presented in the original Hung et al. paper, it could be argued that there might be still contamination of myeloid cells (CD38+) [15], leukocytes (CD50+) [16], and hematopoietic progenitors (HLA-DRdim+, CD90+) [17] cells in the SS fraction. However, these findings were not discussed in sufficient depth by the authors. Based on the current literature [11,18], the absence of these cells in a pure stem cell culture is a conditio sine qua non to confirm efficacious purification of stem cells out of inhomogeneous cell population. In any proposed stem cell isolation process, the efficacy of the separation procedure can be demonstrated when the proportion of these "contaminating" cells is minimized or even depleted. Thus Hung et al. may need to show in future studies that there is a significantly lower proportion of contaminating marrow cell types among the SS cells compared to the LP cells and the unseparated cell population. The efficacy of this stem cell separation procedure can therefore be demonstrated if a significantly lower proportion of contaminating cells is observed. With sufficiently low contaminating cells, the additional step of depleting such "contaminating" cells via cell sorters (using cell surface markers like STRO-1 and CD105) may then be avoided. In addition, the use of 3 µm might filter out residual amounts of red blood cells (RBCs) and platelets (2–4µm) that are not entirely removed by the Percoll fractionation method. Though RBCs are 7.5 µm in size, they are able to deform and squeeze through 2 µm capillaries (cellular sizes referenced from [14]).
In conclusion, it would be meaningful to evaluate and compare the differentiation potential and surface markers for the lower plate cells to support the conclusion that the 3-µm sieve has separated the stem cells, leaving them on the upper plate. If successful, further proof from in vivo functionality studies on the unpurified, suspect-purified, and suspect-depleted fractions may further strengthen the usefulness of this technique. It is only with such supporting data that the method proposed by Hung et al. could claim to be a reliable, inexpensive, user-friendly, and novel strategy to separate stem cells for further characterization and finally for application in regenerative medicine.
ACKNOWLEDGMENTS
We acknowledge the scientific insights provided by the reviewers of this letter.
REFERENCES
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