Reflection and observation: cell-based screening failing to detect HBV in HUMSCs derived from HBV-infected mothers underscores the importance of more stringent donor eligibility to reduce risk of transmission of infectious diseases for stem cell-based medical products

Abstract

Background: In cell-based therapy, the transmission of communicable diseases imposes a substantial threat to recipients. In this study, we investigated whether cell-based screening could detect hepatitis B virus (HBV) in human umbilical cord-derived mesenchymal stem cells (HUMSCs) isolated from HBV-infected donors to understand the susceptibility of HUMSCs to HBV infection.

Methods: HBV assay was performed in HUMSCs derived from healthy and HBV-infected donors with enzyme-linked immunosorbent assay (ELISA), fluorescence quantitative PCR (FQ-PCR) assay, and droplet digital PCR (ddPCR) assay. Further, HBV DNA was assayed in HUMSCs derived from healthy donors after incubation with human sera containing a high titer of HBV using FQ-PCR.

Results: HBV antigen/antibody and DNA failed to be detected using ELISA, FQ-PCR, and ddPCR. After incubation with HBV infection sera, HBV DNA could be detected, but below the valid titer of the assay kit. The HBV DNA levels in HBV-incubated HUMSCs gradually decreased with medium change every 2 days and then significantly decreased, not even detected after passage.

Conclusions: The current cell-based screening methods could not detect HBV in HUMSCs derived from HBV-infected donors, indicating the importance of more stringent donor eligibility to reduce the risk of transmission of communicable diseases in cell-based therapy. To solve the problem of an occult HBV window period in donor eligibility determination, we recommend that the donors undergo another HBV serological test 3 months after the first serological communicable disease screening.

Keywords: Cell-based therapy; Clinical screening assay; Droplet digital PCR; Hepatitis B virus; Human umbilical cord mesenchymal stem cells; Serological test.

Fig. 1

Characterization of HUMSCs. a HUMSCs derived from a healthy donor (No. 3) positively expressed CD44, CD73, CD90, and CD105, but negatively expressed CD11b, CD19, CD34, CD45, HLA-DQ, and HLA-DR by flow cytometry analysis. b Morphology of HUMSCs under light microscope. Scale bars = 500 μm. c, d Oil red O staining and Alizarin red-S staining showed HUMSCs were induced into adipogenic and osteogenic cells, respectively. Scale bars = 100 μm

Fig. 2

Amplification curve of HBV diluted standard. The value of ΔRn indicates the amount of probe degradation during PCR, which is the amount of PCR product. Valid detection limit of HBV PCR fluorescence quantitative detection kit was 100 IU/ml

Fig. 3

HBV DNA detection in HUMSCs after incubation with human sera with HBV using FQ-PCR. a HBV DNA detection in HUMSCs without medium change and passage after incubation with human sera from HBV-infected donor. b HBV DNA detection in HUMSCs with medium change every 2 days. HBV DNA levels gradually decreased in cell lysate and medium from day 0 to 8. c HBV DNA detection in HUMSCs with passage. In cell lysate, HBV DNA level in P3 HUMSCs significantly higher that than in P4 HUMSCs, and failed to be detected in P5–P7 HUMSCs. In culture medium, HBV DNA detected only in P3. HBV hepatitis B virus, * represents p < 0.05 and p < 0.05 was considered statistically significant