Cytotherapy. 2014 Apr;16(4):560-5. doi: 10.1016/j.jcyt.2013.10.009.

Recovery of CD45(-)/Lin(-)/SSEA-4(+) very small embryonic-like stem cells by cord blood bank standard operating procedures.

Chang YJ1, Tien KE1, Wen CH1, Hsieh TB1, Hwang SM2.
  • 1Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan.
  • 2Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. Electronic address: hsm@firdi.org.tw.

 

 

Abstract

BACKGROUND AIMS:

Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines.

METHODS:

The recovery of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction.

RESULTS:

CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing.

CONCLUSIONS:

The rare sub-population of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens.

KEYWORDS: Hespan sedimentation; cell recovery; cord blood bank standard operating procedures; very small embryonic stem cells

PMID: 24364909

 

Supplement:

Since the first umbilical cord blood transplant was performed to treat Fanconi anemia in 1989, more than 30,000 patients worldwide have had umbilical cord blood transplants over the last 20 years. Cord blood (CB) has become a valuable source of stem cells therapy, and both private and public cord blood banks have actively developed worldwide.

It has been known that there are many kinds of stem cells can be found in CB, such as hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), and very small embryonic-like (VSEL) stem cells (1). VSEL stem cells, a rare population in CB or bone marrow, display small size (3-5 μm) and high nuclear-cytoplasmic ratio, express some pluripotency markers (Oct-4 and SSEA4 etc.) and differentiate into all three germ-layers in vitro (2). Because of the difficulty in isolation and cultivation of VSEL stem cells, relatively less information on VSEL stem cells is currently available.

Recent article claimed that VSEL stem cells in cord blood were discarded with red blood cells (RBCs) by Ficoll-Plaque gradient centrifugation during cord blood banking (3). However, most cord blood banks use Hespan as enhance sedimentation of RBCs for rouleaux formation by the protocol– Cord Blood Transplantation Study: cord blood bank standard operating procedures (COBLT SOP) (4). Here, we tried to check if the VSEL stem cells could be preserved after COBLT SOP processing. Each CB unit was divided into two parts: the unprocessed (whole blood) part and the COBLT SOP processed part. Then, the frequency of VSEL stem cells, defining as CD45/Lin/SSEA-4+, was analyzed respectively. After estimating the total number of VSEL stem cells within each part, we concluded that over 80% VSEL stem cells could be reserved from CB after COBLT SOP processing.

The mononuclear cells (MNC) are separated from RBCs by Ficoll-Plaque gradient centrifugation based on the density difference, and VSEL cells might be discarded with RBCs due to the similar cell density (Fig.1A). However, the total nuclear cells (TNCs) are collected from the buffy coat by Hespan-added RBC sedimentation, and VSEL stem cells can be recovered in TNCs (Fig.1B). Our finding suggests that UCB units cryopreserved by COBLT SOP in cord blood banks should retain a significant proportion of CD45/Lin/SSEA-4+ VSEL stem cells.

 

Fig.1

Figure 1: Distribution of cord blood components after Ficoll-Plaque processed (A) and Hespan sedimentation (B).

 

Acknowledgments:

This work was supported by the Ministry of Economic Affairs, Taiwan, (101-EC-17-A-01-04-0525) and SinoCell Technology, Taiwan.

 

References:

  1. Ali, H. and Al-Mulla, F. (2012) Defining umbilical cord blood stem cells. Stem Cell Discovery, 2, 15-23.
  2. Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak J, Kucia M. Very small embryonic-like stem cells: characterization, developmental origin, and biological signi fi cance. Exp Hematol. 2008;36:742 e 51.
  3. Bhartiya D, Shaikh A, Nagvenkar P, Kasiviswanathan S, Pethe P, Pawani H, et al. Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy. Stem Cells Dev. 2012;21:1 e 6.
  4. Fraser JK, Cairo MS, Wagner EL, McCurdy PR, Baxter-Lowe LA, Carter SL, et al. Cord Blood Transplantation Study (COBLT): cord blood bank standard operating procedures. J Hematother. 1998;7:521 e 61.

 

 

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