Protocol for peripheral blood stem cell isolation needs further studies

To the Editors,

The article titled: Molecular characterisation of human peripheral blood stem cells, which was written by Ab Kadir R et al, cultured and characterised stem cells from human peripheral blood.¹ It is well known that peripheral blood contains mature blood cells and poor in progenitors and stem cells, unless the stem cells or progenitors are mobilized using granulocyte macrophage colony stimmulating factor (GM-CSF). Previous attempts to culture stem cell-like cells or progenitor cells from unmobilised peripheral blood used specific growth factor containing medium,² or  did enrichment before culturing using monoclonal antibody-linked magnetic beads.³

 

Therefore, the success of the authors in culturing the ‘so called’ stem cells from unmobilised peripheral blood without enrichment and using merely newborn calf serum supplemented  MEM is of great value. However, the protocol did not contain enough information that is required when other researcher want to repeat the study to get a prove.

 

Missing information are:

• Number of cell seeding per well
• Whether the morphology of the plastic adherent cells (that is supposed to be mesenchymal stem cell/MSC) after 14 days is homogenously fibroblastic
• Whether the plastic adherent cells in primary culture are forming colonies or directly form cell network and grow in monolayer
• The success rate of this protocol in to grow plastic adherent cells
• Whether the plastic adherent cells can be subcultured and untill how many times (if they are subcultured)
• Whether the plastic adherent cells from passage 1 and so on can live to attain confluency, and the number of cell seeding and  time needed to become confluence (if they are subcultured)
• The success rate of subculture (if they are subcultured)

 

Number of cell seeding is very important, because of the low amount of stem cells in peripheral blood. When the amount of stem cells is low, such as in umbilical cord blood, seeding density should be increased. A study used a cell density of 10⁶/cm² when using Ficoll separated mononuclear cells, and hence did not get mesenchymal stem cells that can pass passage-1,⁴ though another study got a 30% success rate. On the other hand, when the stem cells are abundant such as in adipose tissue, for primary culture, a seeding density of 50.000 cells in 25 cm2 TC flask is enough to yields enough colonies to be subcultured.⁵

 

Morphology of MSC is fibroblastic. In case of adipose tissue derived plastic adherent cells, primary cultures may yield colonies when the seeding density is low, or the cells may directly form a monolayer when the seeding density is high. However, Figure 1b¹ is not clear enough to judge whether the cells are forming a colony, as the magnification is too high, and the area is not wide enough to see the boundary of the colonies. Further, in Figure 1b we can see part of an adherent mononucleated large rounded cell that is clearly non fibroblastic. This large rounded cell can be seen clearly (as a whole cell) in another article written by the same group of authors, ⁶ and I supposed to be a monocyte/macrophage lineage, as it is similar in morphology to monocyte/macrophage lineage from umbilical cord blood (our unpublished result).

The 14-day culture of plastic adherent cells after separation from floating cells are intended to deplete contaminating cells such as granulocytes and monocytes.¹ Indeed, mature monocytes will be eliminated after 5 days. However, there is posibility of the presence of contaminating monocyte/macrophage progenitors. In case of umbilical cord blood MSC, monocyte/macrophage lineage tend to adhere to plastic more than the fibroblastic cells, and in subsequent passage tend to form osteoclastic-like cells that overgrow the fibroblastic cells.⁴ Moreover, monocyte-derived subsets from peripheral blood may appear fibroblastic, and can be differentiated into various kinds of cells under proper induction medium.⁷

Finally, according to a consensus, the characterization of mesenchymal stromal cells, which is recently called MSC, should minimally show that more than 95% of the cells bear CD 105, CD 90 and CD 73; further, MSCs should have the abilities to differentiate into 3 lineages i.e. adipocyte, chondrocyte, and osteocyte lineages.⁸ This study only showed the ability to differentiate into osteocyte lineage, and used CD 105 expression to characterize the plastic adherent cells.¹ Expression study using RT-PCR may yield positive result even from a single MSC, therefore the criteria of more than 95% can not be fulfilled.

Therefore, the protocol needs to be further studied, whether the fibroblastic adherent cells are really MSCs.

References

1.

Ab Kadir R, Ariffin SHZ,Wahab RMA, Senafi S. Molecular characterisation of human peripheral blood stem cells. S Afr J Sci. 2012;108(5/6):7 pages.  Art.#939

2.

Filippone C, Franssila R, Kumar A, Saikko L, Kovanen PE, Söderlund-Venermo M, et al. Erythroid Progenitor Cells Expanded from Peripheral Blood without Mobilization or Preselection: Molecular Characteristics and Functional Competence. PLoS ONE 2010; 5(3): e9496.

3.

Abuljadayel IS. Induction of stem cell-like plasticity in mononuclear cells derived from unmobilised adult human peripheral blood. Curr Med Res Opin. 2003;19(5):355-75.

4.

Kawasaki-Oyama RS, Braile DM, Caldas HC, Leal JCF, Goloni-Bertolo EM, Pavarino-Bertelli EC, et al. Blood mesenchymal stem cell culture from the umbilical cord with and without Ficoll-Paque density gradient method. Rev Bras Cir Cardiovasc. 2008; 23(1): 29-34.

5.

Pawitan JA, Bustami A, Damayanti L, Antarianto R, Swantari NM. Effect of adipose tissue processing procedures in culture result: A preliminary study. Med J Indones 2011;20(1):15-9.

6.

Ab Kadir R, Ariffin SHZ,Wahab RMA, Kermani S,Senafi S. Characterization of Mononucleated Human Peripheral Blood Cells. ScientificWorldJournal 2012; 2012;8 pages, Article ID 843843.

7.

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society forCellular Therapy position statement. Cytotherapy. 2006;8(4):315–7.

8.

Zhao Y, Glesne D, Huberman E. A human peripheral blood monocyte-derived subset acts as pluripotent stem cells. Proc Natl Acad Sci USA 2003;100: 2426-2431.

 

 

Jeanne Adiwinata Pawitan

Department of Histology, Faculty of Medicine, Universitas Indonesia

Jakarta, Indonesia

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