PB-MNCs from four male patients were collected before A-TAH implantation (T0) and after implantation at one month (T1), between two and five months (T2), and then between six and twelve months (T3). As the A-TAH hybrid membrane has been described as being endothelialized after several months of implantation (7), we decided to explore the phenotype of circulating stem cells after implantation in order to hypothesize the cell origin of these newly formed endothelial tissues. Supervised analysis of flow cytometry data confirmed the presence of the previously identified Lin-CD133+CD45- and Lin-CD34+ with different CD45 level intensities. Lin-CD133+CD45- and Lin-CD34+CD45- were assumed to contain VSELs and were not modulated during the period studied here. The statistical analysis showed that, among the three populations of CD34+ only the Lin-CD34+CD45dim was significantly increased one month after A-TAH implantation in contrast to pre-implantation level (p = 0.01), regardless of the expression of CD133 or c-Kit. Indeed, Lin-CD34+CD45dim population could be sub-divided into four categories according to the positivity of CD133 and c-Kit. When CD133 or c-Kit were positive, we always observed a significant increase in stem cells after implantation, whereas there was no significant difference after A-TAH implantation in the Lin-CD34+CD45dimCD133−c-Kit− population. The data for the resulting clusters are visualized on a UMAP plot in Figure 1 showing all single cells of the live Lin−and CD34+events selected from down sampled files concatenated at T0 and T1. The algorithm proposed seven clusters. As demonstrated in Figure 1A, three of the seven clusters evidenced are upregulated in T1, in contrast to T0: clusters 7, 4, and 2. In Figure 1B, analysis demonstrated that the increase concerned the three CD45dim clusters, confirming our results presented in Table 2. Thus, using a flow cytometry approach, we showed a significant mobilization of Lin-CD34+CD45dim in peripheral blood one month after A-TAH implantation. We recently described a progressive endothelialization of the bioprosthetic hybrid membrane of the A-TAH that could be at the origin of its acquired hemocompatibility (7). As there is no physical connection between the internal membrane of the device and the patient’s blood vessels, the source of these neo-endocardial cells in the A-TAH shall come from the circulating blood. Thus, the aim of this study was to identify by conventional flow cytometry approaches stem cells in blood that could be mobilized and could give rise to newly formed endothelial cells on the hybrid membrane of the A-TAH.
In the past years, CD34+ cells emerged as the most convincing cell type among those that have been evaluated for their use in cell-therapy trials and as biomarker of cardiac disease (10). CD34+ hematopoietic stem cells with the CD45dim phenotype have been proposed as a source of extra hematopoietic cells like cardiomyocytes for example (18), although this has been controversial (19). However, in human adults, we don’t know with certitude the stem cell at the origin of ECFCs. Indeed, it is now admitted that ECFCs are the main human post-natal vasculogenic cells (8). ECFCs have been described to grow from circulating CD34+ cells present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. CD34+ and CD34− ECFCs have different angiogenic properties and CD34 expression in ECFCs could be related to a specific state of endothelial phenotype (20). Their origin has been proposed in CD45 negative cells (21) but subtype of CD34 involved in ECFC differentiation is unclear (22). CD34+ cell sub-populations may be derived from VSELs. VSELs were first identified as CD45 negative cells and characterized by their very small size (3-5 μm in diameter) in murine and human bone marrow (5-6 μm in diameter) (14). VSELs are mobilized into peripheral blood in response to injury following acute myocardial infarction (23) or critical leg ischemia (15) and we previously demonstrated that these cells trigger post-ischemic revascularization (15). Others and we have also shown VSELs ability to differentiate into endothelial cells (15, 24-26). Human VSELs have been described expressing CD133, but some description of human CD34+-VSELs have been done and their vascular differentiation ability confirmed (27). CD34+-VSELs can regenerate damaged organs and may solve the problems inherent in the use of controversial embryonic stem cells or induced pluripotent stem cells indeed. In our study, we did not include any size beads. However, when back gating our populations, we can assume that Lin-CD133+CD45- cells are only small sized cells compatible with VSELs phenotype. In contrast, Lin-CD34+CD45- and Lin-CD34+CD45dim are a mix of small and large sized CD34+ cells. Lin-CD34+CD45dim of small size has never been specifically studied in terms of multipotent differentiation ability. Thus, we observed the mobilization of a CD34+ population with CD45dim expression while the CD45neg population was not mobilized. This CD45dim population contained various-sized cells. Further study needs to evaluate the ability of CD45dimCD34+ cells of small and “normal size” to give rise to endothelial cells in vitro and in vivo and validate the origin of newly formed endothelial cells on top of A-TAH hybrid membrane.
All in all, bioprosthetic A-TAH implantation allowed us to evidence the mobilization in peripheral blood of Lin-CD34+CD45dim stem cells that could be at the origin of the endothelial recovery. In order to organize new cell-therapy trials or determine the cells at the origin of endothelial lineage in vivo further studies need to appreciate the size of stem cells that are mobilized and able to build vessels. This topic of adult stem cells at the top of the hierarchy of endothelial lineage requires research on stem cells in peripheral blood in other cardiovascular mobilization situations, especially organ replacement requiring cell recolonization. New multidimensional proteomic approach by flow, imaging, or mass cytometry associated with bioinformatic analysis may help to ameliorate the screening of stem cells involved in the vasculogenic process.