First-in-human clinical study of an embryonic stem cell product for urea cycle disorders

Abstract

Background: This study assesses the safety and efficacy of hepatocyte-like cell (HLC) infusion therapy derived from human embryonic stem cells as bridging therapy for neonatal-onset urea cycle disorders (UCD). The research includes both preclinical and clinical evaluations to determine the feasibility of HLC infusion as a therapeutic option for safer pediatric liver transplantation.

Methods: Preclinical studies were conducted to validate the safety, biodistribution, and ammonia metabolism capabilities of HLCs using SCID mice models of UCD and extensive animal studies. In the clinical trial, five neonates with UCD received HLC infusions, intending to maintain metabolic stability and exceed a target weight of over 6 kg, which is considered necessary for safer liver transplantation.

Results: Preclinical studies demonstrated that HLCs successfully engrafted in the liver without adverse migration or tumor formation and effectively elongated survival. Clinically, all five neonates exceeded the target weight of 6 kg while maintaining metabolic stability and successfully bridging to transplantation. Post-transplantation follow-up revealed stable growth, metabolic control, and no neurological complications.

Conclusions: The combined preclinical and clinical findings support HLC infusion as a viable bridge therapy for neonates with UCD, providing metabolic support to achieve safer weight thresholds for transplantation. While promising, careful monitoring remains essential, particularly for potential complications such as thrombus formation.

Trial registration: jRCT, jRCT1090220412. Registered on 27 February 2019, https://jrct.niph.go.jp/en-latest-detail/jRCT1090220412 (originally registered in JMACCT (JMA-IIA00412)).

Keywords: Ammonia removal; Cell-based therapy; Embryonic stem cells (ESCs); Hepatocyte; Hyperammonemia; Liver transplantation; Neonatal-onset; Regenerative medicine; Urea cycle disorders (UCD).

Fig. 1

Characterization of HLCs. A Phase-contrast photomicrograph of undifferentiated ESCs (left: pHAES-WCB, Bar: 1.0 mm) and HLCs (right: HAES, Bar: 50 µm). B Histology of HLCs in iPGell. H.E. stain. Bar: 100 µm. C Ultrastructural analysis of HLCs. Left panel: HLCs showed polarity with microvilli on the apical membrane. HLCs had tight junctions and desmosomes between the cells. Bar: 10 µm. Middle panel: HLCs exhibited glycogen β-particles and had abundant mitochondria in the cytoplasm. Bar: 1.0 µm. Right upper panel: HLCs had microvilli at bile canaliculi and tight junctions between cells. Right lower panel: HLCs had large amounts of glycogen particles in the cytoplasm. N: nucleus, Nu: nucleus, Tj: tight junction, D: desmosome, MV: microvillus, G: golgi body, M: Mitochondria, BC: bile canaliculus. D Glycogen accumulation in HLCs. Periodic acid–Schiff (PAS) stain. Bar: 200 μm. E Immunohistochemistry of HLCs with an antibody to E-cadherin. Bar: 50 μm. F Immunohistochemistry of HLCs with an antibody to AFP. Bar (upper panel): 100 μm, Bar (lower panel): 50 μm. G Immunohistochemistry of HLCs with an antibody to cytokeratins (AE1/3). Bar: 100 μm. H, I Time-course analysis of ammonia concentration was measured in culture media of undifferentiated ESCs (H) and HLCs (I). J Ammonia metabolic activity of undifferentiated ESCs and HLCs was summarized. K Effect of human hepatocyte-like cell treatment on the survival of SCID-OTCD mice compared with human cryopreserved hepatocytes

Fig. 2

Embolization using a vascular plug under IVR. A Macroscopic view of AmplatzerTM Vascular plug used for embolization. To avoid systemic circulation of HLCs, an open ductus venosus is closed before injection. B Embolization of ductus venosus. Ductus venosus is closed 15–20 days after birth [26]. The ductus venosus was patent in Cases 1 and 2 and thus was embolized before the transplantation. C AmplatzerTM Vascular plug in the explanted liver. D High-power view of AmplatzerTM Vascular plug (C, dotted rectangle)

Fig. 3

Clinical courses of HLC treatment. A–E Profiles of blood ammonia levels of the patients with urea cycle disorder from the onset of the disease to liver transplantation. Arrowheads indicate the time of HLC infusion (HLC inf), and the numbers in parentheses are the ages and days of transplantation. Arrows indicate the time of liver transplantation. LDLT: living donor liver transplantation (A, D, E), DDLT: deceased donor liver transplantation (B, C). Values at marked hyperammonemia seizures are shown in the graph. Asterisks indicate major clinical episodes; enterocolitis (A), enteritis (B), hypertrophic pyloric stenosis, and pyloromyotomy (B, D). F A list of human hepatocyte-like cell treatments. *1 CTLN1: citrullinemia type 1, CPS1D: carbamoyl phosphate synthetase 1 deficiency, OTCD: ornithine transcarbamylase deficiency, *2 HLCT: hepatocyte-like cell treatment. G Clinical course after hepatocyte-like cell treatment and liver transplantation. *1 CTLN1: citrullinemia type 1, CPS1D: carbamoyl phosphate synthetase 1 deficiency, OTCD: ornithine transcarbamylase deficiency, *2 HLCT: hepatocyte-like cell treatment, *3 LT: liver transplantation, *4 LDLT: living donor liver transplantation, DDLT: deceased donor liver transplantation, GRWR: graft-recipient weight ratio, *5 POD: postoperative day

Fig. 4

Macroscopic and microscopic view of the explanted liver in Case 4, OTCD. A–E Macroscopic anterior view of livers. The patients’ livers that received ESC-derived hepatocytes showed a smooth and normal surface in all cases upon macroscopic examination, despite a functionally disabled urea cycle, just before they were resected for transplantation. No discoloration or ischemic changes were detected; no tumors were formed. A Case 1: CTLN1, B Case 2: CPS1D, C Case 3: OTCD, D Case 4: OTCD, E Case 5: CPS1D. F Macroscopic view of the superior surface of the explanted native liver in Case 4. G A white thrombus (white circle) in the umbilical portion of the portal vein upon examination of the cut surface of the explanted native liver in Case 4. H Low-magnification histopathological analysis of the thrombus in Case 4. I Chondrogenesis observed within the embolus. Upper right panel: chondrogenesis. Lower left panel: inflammatory cell infiltration in Case 4. J Contrast-enhanced CT scan in Case 4. Lt. PV: left portal vein, UP: umbilical portion. K Evaluation of portal vein flow 2 months after the hepatocyte transplantation in Case 4. L Evaluation of portal vein flow at three months post-hepatocyte transplantation, or one-month pre-liver transplantation in Case 4