Isolating Bone Marrow Stem Cells Using Sieve Technology

Abstract

The ability and efficiency of various techniques to purifyadult stem cells from a heterogeneous cell population is animportant determinant for the successful characterizationand application of stem cells. Strategies employed to isolateor purify such cells are numerous,and these include sortingof cells according to specific cell surface markers usingeither fluorescence [ 1] (the current gold standard in purify-ing stem cells) or immunomagnetic technology [ 2],exploit-ing the differential plating efficiencies of stem cells on cul-ture 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 sepa-rate stem cells from a heterogeneous population.Hung et al. described plating Percoll gradient separatedbone marrow stem cells on double-decker culture plates(Transwell,Corning,NY) where the top plate was fabricatedwith 3- µm pores. Size-sieved (SS) postcultured cells on theupper plate were described to be fibroblastic in morphologyand large in size,while the lowere plate (LP) cells were smalland polygonal. A series of assays characterizing the differen-tiation potential, proliferation, and a selected group of sur-face markers was reported,but only on the SS cells. Hung etal. suggested that the 3-µm pores separated the stem cellsfrom the heterogeneous cell population. The true advantageand attractiveness of the proposed strategy would be its costeffectiveness, straightforward and user-friendly protocol.However, it would be more convincing to the scientificcommunities of stem cell and tissue engineering if the LPcells were also characterized and shown not to possess thesame differentiation potential and characteristics. Thiswould be important before one can arrive at the conclusionthat 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 proposedstrategy. The greater the efficacy, the more localized stem-ness would be on SS cells and the less on LP cells. Conse-quently, this would mean that the SS to LP data justify andreflect the efficacy of the procedure. Reporting SS cells ashaving stem cell characteristics may not be adequate becausewithout sieve separation, the multipotency of bone mar-row–derived stem cells have already been reported [6–10],thus leading us to expect some degree of stem cell character-istics with the SS cells anyway. Confirming the stem-ness ofa stem cell population must precede its characterization.Without such confirmation,any conclusions arising from thecharacteristics of the purported stem cell population wouldbe incomplete, and the derived clinical applications wouldhave profound repercussion throughout the field. Ideally,research should assess stem-ness in terms of cellular self-renewal ability–the differentiation of in vitro and in vivo sin-gle-cell clones into cell types of the tissue of origin and atleast one other different tissue cell type [11].In addition, the characterization of LP cells would alsoprovide some insights to stem cell biology. The presupposi-tion of the proposed strategy is that stem cells in suspensionwould be larger than 3 µm and would thus remain in the upper