Identification and quantification of very small embryonic-like (VSEL) stem cells in fresh umbilical cord blood

The present study aimed to identify, quantify and compare the quantities of very small embryonic-like (VSEL) stem cells in the two main fractions of fresh umbilical cord blood (UCB). To do this, UCB obtained during caesarean section underwent gradient centrifugation. Nucleated cells isolated from the two main fractions – the mononuclear cell layer above the gradient, and the red blood cell pellet below the gradient – were immunofluorescently labelled to identify the VSELs (CD45−/CD133+/SSEA4+). These two cell fractions were then analysed via multiparameter sorting on a flow cytometer and the quantities of positively stained cells were compared.


Introduction
Human hematopoietic sources are known to harbour various adult pluripotent stem cell populations [1].One such cell typevery small embryonic-like (VSEL) stem cellswas first isolated and characterised in the murine bone marrow [2,3] and later identified in human umbilical cord blood [4].This population has since gained significant interest due to its ability to give rise to all three germ cell layers combined with its lack of tumourogenicity [5,6,7].It has therefore been proposed as a promising cell source for tissue regeneration [8].
Nonetheless, there are numerous challenges and controversies surrounding these cells.Firstly, there are reported to be a rare population and there is a lack of consensus regarding these cells' location (blood layer during processing) and quantities in umbilical cord blood (UCB).Moreover, there is heterogeneity in the way different authors define and immunophenotype the VSEL population depending on the hematopoietic source and technique used [1,4,6,7,9,10].
The objective of the study was therefore to identify, quantify and compare the quantities of VSEL stem cells in the two main cell fractions of fresh UCB.

Patients and UCB processing
This study was performed in accordance with the Declaration of Helsinki.Collection of human tissue samples for this study was approved as part of the study protocol.This human study was approved by Nadezhda Women's Health Ethics Committee.All adult participants provided written informed consent to participate in this study.
Pregnant female patients with no pregnancy complications, infectious, autoimmune, endocrinological or oncological condition were identified through hospital records and invited to participate in the study.
After signing informed consent, fresh UCB was needle aspirated from 5 eligible patients during scheduled caesarian section delivery.Blood samples were collected in sterile 8.5 ml Vacutainer tubes with acid citrate dextrose as anticoagulant (BD 366645).The samples were processed via standard gradient centrifugation protocol.In brief, blood was diluted 2-fold with PBS and overlayed on 1.077g/ml Pancoll.It was then centrifuged at 400G for 25 min.Cells from the mononuclear cell (MNC) fraction (above the Pancoll) and red blood cell (RBC) pellet (blow the Pancoll) were collected in separate tubes.The RBC layer underwent erythrocyte lysis with a hypotonic buffer (Roche, cat no.11814389001).
A schematic overview of the experimental protocol and gating strategy can be seen in Figures 1 and  2, respectively.

Discussion
The existence of VSELs has been heavily debated ever since they were first reported [8].The main reason for the scepticism surrounding these cells is that there is a lack of standardisation regarding the markers used for phenotyping VSELs.Some heterogeneity stems from the different hematopoetic source in which they have been studied (bone marrow, peripheral blood, umbilical cord blood) [1].Nonetheless, most protocols use principal characteristics of VSELs to identify themsmall size, lack of granules, lack of expression of the common leukocyte antigen (CD45) and other other hematopoietic lineage markers (Lin-), expression of general "stem-ness" marker like CD34 or CD133 and embryonic markers like Oct-4, stage-specific embryonic antigens (SSEAs), CXCR4, Sox2 or Nanog [1,4,6,7,9,10,11].The present study employed common markers reported by other authors for identification of VSELs specifically in umbilical cord blood, namely CD133 and SSEA4 [4].From a quantitative standpoint, there also seems to be heterogeneity in terms of the units used by different authorssome report the VSELs quantity as cell count /ml of whole blood [10], others -number of sorted cells [11].However, most studies, including this one, report percent of nucleated cells, which is arguably an objective metric [1].
In terms of the location of VSELs, some authors have reported that they pass through the gradient during cord processing due to their small size and are mostly found in the RBC pellet [12,13].In contrast to these findings, the present study suggests that VSEL stem cells are predominantly present in the MNC layer of umbilical cord blood.Since standard UCB processing protocols for biobanking isolate the leukocyte fraction, these cell types are likely also retained during cryopreservation.Nonetheless, a portion of them was also found in the lysed RBC pellet like previously suggested.To increase yield, it might be useful to retain and process that fraction which is usually discarded.
When it comes to potential applications, VSELs have been shown to be able to differentiate into neural, bone, cardiac and lung tissue [5,6,7] and these may be used in preclinical drug testing.Nonetheless, in order to become suitable for clinical application in their respective areas of regenerative medicine, there is a requirement for larger quantities of these otherwise scarce cells.Recent successful efforts for large-scale reproducible expansion of CD34+ cells have reported an increased fraction of Lin-CD45-CD133+ VSELs [14].Although promising, further research into methods for efficient VSEL culture and expansion is necessary.
Another active area of research in VSELs is reproduction.VSELs have been reported in human and mouse reproductive organs such as testis [15], ovary [16] and uterus [17].It has also been shown that VSELs are able to differentiate into gametes [18,19,20].Moreover, these cells have been shown to be resistant to oncotherapy, mainly because of their stability during quiescent state [21].Therefore, the characteristics of these cells make them a suitable tool for assisted reproduction and oncofertility preservation.

Conclusion
In summary, VSELs are a promising stem cell type that resemble embryonic stem cells in terms of their pluripotent properties, and are easily accessible since they are present in adult tissues and cord blood.They are found in both the mononuclear layer as well as in the RBC pellet fraction after processing.Future studies should focus on efficient isolation, culture and cell expansion methods for VSELs before they can be tested for the clinical value in regenerative medicine.

Figure 1 .
Figure 1.Diagram illustrating the experimental design of the study.MNCmononuclear cells, RBCred blood cells.Created with BioRender.com.

Figure 2 .
Figure 2. Gating strategy for identification and quantification of umbilical cord blood-derived VSELs by FACS.