Industrialized GMP Production of CD34+ Cells (ProtheraCytes®) at Clinical Scale for Treatment of Ischemic Cardiac Diseases Is Feasible and Safe

Abstract

Regenerative medicine now needs to pass a crucial turning point, from academic research to the market. Several sources/types of cells have been experimented with, more or less successfully. CD34⁺ cells have demonstrated multipotent or even pluripotent capacities, making them good candidates for regenerative medicine, particularly for treating heart diseases. Strongly encouraged by the results we achieved in a pilot study using CD34⁺ stem cells in patients with poor-prognosis acute myocardial infarcts (AMIs), we soon began the development of an industrialized platform making use of a closed automated device (StemXpand^®) and a disposable kit (StemPack^®) for the large-scale expansion of CD34⁺ cells with reproducible good manufacturing practice (GMP). This scalable platform can produce expanded CD34⁺ cells (ProtheraCytes^®) of sufficient quality that, interestingly, express early markers of the cardiac and endothelial pathways and early cardiac-mesoderm markers. They also contain CD34⁺ pluripotent cells characterized as very small embryonic-like stem cells (VSELs), capable of differentiating under appropriate stimuli into different tissue lineages, including endothelial and cardiomyocytic ones.

Keywords: CD34+ cells; Cell expansion; Heart diseases; Industrialization; ProtheraCytes®; Regenerative medicine; VSELs.

Fig. 1

The StemXpand^® cell expansion platform: V1 clinical version with one incubator and production kit (left); and the V2 clinical version (on process) comprising 5 incubators and an aseptic cassette replacing the previous kit of production (right)

Fig. 2

Heat map representing the differential expression of cardiac and endothelial very early markers detected in ProtheraCytes^® versus native CD34⁺ cells at day 0 from AMI patients at day 0. Genes are grouped into three categories, including mesoderm, cardiac mesoderm markers (A) and endothelial markers (B) and cardiac markers (C). Green boxes (A, B, C) highlight HEY1, NCAM1, MESP1, ANGPT2, FLT1, ESAM, ALCAM, MYL4, TNNT1 and PDLIM5. Native CD34⁺ SC are represented in blue (n = 7), the ProtheraCytes^® samples are in purple (n = 8). (D) Heatmap showing the main cardiac, mesoderm and endothelial genes induced in ProtheraCytes^® by comparison to CD34⁺ cells expressed as fold change (FC); the color scale is represented at the bottom

Fig. 3

Heat map representing the differential expression of cardiac and endothelial very early markers detected in ProtheraCytes^® versus native CD34⁺ cells at day 0 from Healthy donors at day 0. Genes are grouped into three categories, including mesoderm, cardiac mesoderm markers (A) and endothelial markers (B) and cardiac markers (C). In (D) Heatmap showing the main cardiac, mesoderm and endothelial genes induced in ProtheraCytes^® by comparison to native CD34⁺ cells expressed as fold change (FC); the color scale is represented at the bottom

Fig. 4

The stem cells and endothelial progenitor cell markers detected in ProtheraCytes^® by flow cytometry from AMI patients (n = 12) and controls (n = 4)

Fig. 5

Representative experiment showing, the Lin⁻CD34⁺CD45⁻CD133⁺ VSELs presence in control on day 0, in day 9 expanded CD34⁺ SC before immunoselection and in purified ProtheraCytes^® as detected by flow cytometry

Fig. 6

Representative experiment showing, the Lin⁻CD34⁺CD45⁻CD133⁺ VSELs presence in AMI patients on day 0, in day 9 expanded CD34⁺ SC before immunoselection and in purified ProtheraCytes^® as detected by flow cytometry

Fig. 7

Proposal for the different biological mechanisms involved in CD34⁺ stem cell cardiac regenerative medicine