A model study for the manufacture and validation of clinical-grade deciduous dental pulp stem cells for chronic liver fibrosis treatment

Background Human deciduous pulp stem cells (hDPSCs) have remarkable stem cell potency associated with cell proliferation, mesenchymal multipotency, and immunosuppressive function and have shown beneficial effects in a variety of animal disease models. Recent studies demonstrated that hDPSCs exhibited in vivo anti-fibrotic and anti-inflammatory action and in vivo hepatogenic-associated liver regeneration, suggesting that hDPSCs may offer a promising source with great clinical demand for treating liver diseases. However, how to manufacture ex vivo large-scale clinical-grade hDPSCs with the appropriate quality, safety, and preclinical efficacy assurances remains unclear. Methods We isolated hDPSCs from human deciduous dental pulp tissues formed by the colony-forming unit-fibroblast (CFU-F) method and expanded them under a xenogeneic-free and serum-free (XF/SF) condition; hDPSC products were subsequently stored by two-step banking including a master cell bank (MCB) and a working cell bank (WCB). The final products were directly thawed hDPSCs from the WCB. We tested the safety and quality check, stem cell properties, and preclinical potentials of final hDPSC products and hDPSC products in the MCB and WCB. Results We optimized manufacturing procedures to isolate and expand hDPSC products under a XF/SF culture condition and established the MCB and the WCB. The final hDPSC products and hDPSC products in the MCB and WCB were validated the safety and quality including population doubling ability, chromosome stability, microorganism safety, and stem cell properties including morphology, cell surface marker expression, and multipotency. We also evaluated the in vivo immunogenicity and tumorigenicity and validated in vivo therapeutic efficacy for liver regeneration in a CCl4-induced chronic liver fibrosis mouse model in the final hDPSC products and hDPSC products in the WCB. Conclusion The manufacture and quality control results indicated that the present procedure could produce sufficient numbers of clinical-grade hDPSC products from a tiny deciduous dental pulp tissue to enhance clinical application of hDPSC products in chronic liver fibrosis. Electronic supplementary material The online version of this article (10.1186/s13287-020-01630-w) contains supplementary material, which is available to authorized users.

The hDPSC-products were maintained at 37 ºC with 5% CO2 in a Forma TM CO2 incubator (Thermo Fisher Scientific). The medium was changed twice weekly.
ice-cold FCMB twice, and were analyzed on a FACSVerse TM flow cytometer (BD Bioscience, Franklin Lake, NJ) using the FACSuite TM software (BD Bioscience). The number (percentage) of positive cells was determined by comparison with the corresponding control cells stained with the corresponding isotype-matched antibody, in which a false-positive rate of less than 1% was accepted. The specific antibodies used for flow cytometric analysis are shown in Supplementary Table 4.

Extraction and preparation of total RNA
Cellular and tissue samples were treated with a TRIzol ® reagent (Thermo Fisher Scientific). The RNA extracts were digested with a DNase I (Promega, Madison, WI), and were purified using an RNeasy ® Mini Kit (Qiagen). To check for genomic DNA contamination, the RNA extracts were directly reacted with a primer pair for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sense, 5'-TGAAGGTCGGTGTCAACGGATTTGGC 3'; Antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3') and Quick Taq HS DyeMix (TOYOBO, Osaka, Japan) with a T-100 TM thermal cycler (Bio-Rad Laboratories). The amplification by PCR was as follows; initial activation step (95 ºC, 120 sec) and three-step cycling (95 ºC for 30 sec, 60 ºC for 30 sec, and 68 ºC for 60 sec; 30 cycles) The concentration of human albumin, HGF, interleukin 6, monocyte chemoattractant protein-1, sialic acid-binding Ig-like lectin-9, mouse matrix metalloproteinase 2 (MMP2), mouse MMP9, mouse tissue inhibitors of metalloproteinase 1 (TIMP1), TIMP2, and transforming growth factor beta 1 in the CM of hDPSC cultures, mouse liver tissues, and mouse peripheral serum were analyzed by ELISA using commercially available kits according to the manufacturers' protocols. Biochemical assays for alanine aminotransferase, aspartate transferase, and total bilirubin in mouse serum were analyzed using commercially available kits according to the manufacturers' protocols.
Finally, the results from the immunological and biochemical tests were measured on a Multiscan TM GO microplate spectrophotometer (Thermo Fisher Scientific). The commercially available kits are listed in Supplementary Table 8.

Preparation for histochemistry and immunohistochemistry
Mouse liver tissue samples were treated with 4% paraformaldehyde (Merck) in PBS (pH 7.4; Nacalai Tesque) for 18 hours. The samples were subsequently dehydrated in a graded ethanol bath and were cleaned in xylene. Finally, the samples were embedded in paraffin, and were cut into 6 mm-thick sections. The paraffin sections were dewaxed and were rehydrated for further histochemical and immunohistochemical studies.

Immunohistochemical analysis
Paraffin sections were incubated with 3.0 % H2O2 in ethanol for 30 min at room temperature to inhibit endogenous peroxidase. The sections were then treated with 10% normal mouse or rabbit serum in PBS for 60 min at room temperature. The sections were treated with primary antibodies overnight at 4 °C, followed by treatment with the EnVision+ System (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instruction. Immunohistochemical negative controls were stained with non-immune isotype-matched IgG instead of the primary antibodies. Primary antibodies and isotype-matched antibodies used in immunohistochemical analysis are shown in Supplementary Table 9.   adipocyte-, chondrocyte-, and osteoblast-specific genes. ALT, alanine aminotransferase; AST: aspartic aminotransferase; HGF, hepatocyte growth factor; IL6, interleukin 6; MCP1, monocyte chemotactic protein 1; MMP2, matrix metalloproteinase 2; MMP9, matrix metalloproteinase 9; SIGLEC9, sialic acidbinding Ig-like lectin 9; TGFb1, Transforming growth factor beta; TIMP1, tissue inhibitor of matrix proteinases 1; TIMP1, tissue inhibitor of matrix proteinases 2.