In 2006, as part of a collaboration between Fresenius Medical Care AG & Co. KGaA and the Camussi lab at the University of Turin, Herrera, Bruno, Buttiglieri et al. identified and characterized a population of cells with multiple differentiating capabilities. They named these cells human liver stem cells (HLSCs).

HLSCs are a novel progenitor population found in the liver, and are phenotypically different from oval cells. They do not express hematopoietic stem cell markers, but do express several mesenchymal markers as well as albumin, alfa-fetoprotein, and cytokeratins 8 and 18. This indicates a partial commitment to the hepatic lineage – they are able to differentiate into hepatocytes, endothelial, and osteogenic cells, but not adipocytes.

The production of HLSCs starts with hepatocytes from fresh surgical specimens of donors undergoing hepatectomies. These are cultured and then expanded under stringent conditions to generate an allogeneic master cell bank of HLSCs. From here, we further expand the HLSCs to generate batches for clinical use.

Clinically, the safety of percutaneous intrahepatic administration of HLSCs has already been demonstrated in an investigator-initiated Phase I study (Spada et al. 2019). HLSCs were administered by two percutaneous intrahepatic injections to three patients within the first months of life. All patients completed the study protocol and all safety endpoints were met. Moreover, no donor specific antibodies against HLSCs were detected. Throughout the study all patients remained clinically stable and no metabolic decompensations were registered after HLSC treatment, despite an increase in protein intake (~30 %). Moreover, all patients who received HLSC therapy did not experience any hyperammonemic crises in the time between HLSC injection and the child’s receipt of a transplant.

Additionally, in vivo, HLSCs are able to increase survival by 75% in a SCID mouse model of hepatic failure and contributed to liver regeneration in a SCID mouse model of acute liver injury. This was demonstrated at 7 and 30 days after HLSC administration (Herrera et al, 2013).

Nano-Extracellular vesicles (nEV) are a heterogeneous group of cell-derived membranous structures comprising smaller exosomes and larger microvesicles. nEVs have been shown to facilitate intercellular communication in diverse cellular processes as they carry a cargo of proteins, nucleic acids, lipids, metabolites, and even organelles from the parent cell.

nEVs have recently been identified as a mechanism of cell-to-cell communication. They act as vehicles that transfer biologically active molecules between originator and recipient cells, therefore influencing the phenotype and function of the latter. Stem cell-derived nEVs act as paracrine mediators of stem cell action as they may activate regenerative programs in injured cells and reprogram cancer cells. Several studies suggested that the embryonic/stem cell microenvironment may favour reprogramming of tumour cells toward a more benign phenotype. In vitro and in vivo experiments have shown that stem cell-derived nEVs cells have a role in such modulation of cancer phenotype by the transferral of transcription factors.

We have identified a unique population of nEVs derived from adult HLSCs (HLSC-nEVs). HLSC-nEVs display an average size distribution of 150nm (range 30-250nm) and express several adhesion molecules. We have shown that HLSC-nEVs are internalized by hepatocytes, and that they are able to modulate various physiological processes by a horizontal transfer of specific RNA subsets.

HLSC-nEVs display miRNA-dependent anti-tumor effects in vitro and in vivo:

  • Fonsato et al. (2012) showed that HLSC-nEVs were able to inhibit tumorigenic liver cells’ proliferation and induce apoptosis in a dose dependent manner; that treatment with very high doses of RNase destroyed this effect; and that transfection of HepG2 cells with specific miRNAs mimicked the anti-tumour effects of intact HLSC-nEVs.
  • Mouse models of HepG2 induced tumors showed significant tumor regression after treatment with HLSC-nEVs, with effects also observed on other tumours such as lymphoblastoma and glioblastoma.
  • Tested on human microvascular endothelial cells (HMECs), HLSC-nEVs reduced vessel-like structure formation significantly, compared to MSC-EVs (Lopatina 2019)
  • HLSC-nEVs significantly reduce tumor angiogenesis and induce apoptosis of established tumors in vivo in an in vivo model (subcutaneous implantation of TEC in Matrigel in SCID mice, Lopatina 2019)
  • In renal cancer stem cells (rCSCs), HLSC-nEVs induced a significant degree of apoptosis (Fonsato 2018), as opposed to MSC-derived EVs
  • Fonsato et al. (2018) showed that renal CSCs chemosensitivity to TKIs was enhanced when HLSC-EVs were co-administered or sequentially added after TKIs

HLSC-nEVs are also capable of displaying major regenerative effect:

  • Herrera et al. (Herrera 2010) showed that HLSC-nEVs induced proliferation and apoptosis resistance of human and rat hepatocytes, thereby protecting healthy cells and supporting liver regeneration.
  • The same team demonstrated that HLSC-nEVs shuttle a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis to their intended target cells to facilitate activation of a proliferative program in remnant hepatocytes.
  • In vivo, when HLSC-nEVs were administered to 70% hepatectomized rats, the morphological and functional recovery of the liver was accelerated and apoptosis was reduced (Herrera 2010, Fig 5+7).

Type 1 diabetes (T1DM) is caused by the autoimmune destruction of pancreatic insulin producing cells (β-cells,) and is associated with the development of debilitating macrovascular and microvascular complications. Treatment with exogenous insulin to attain glycemic control does not completely prevent long term complications. Both pancreas and islet transplantation can restore islet function and potentially reduce long-term diabetic complications, but are limited by both donor shortage and need for immunosuppression. While β-cells can be produced from pluripotent stem cells, the approaches are generally complex and highly inefficient. Additionally, β-cells alone do not replicate the activity of intact islets, which contain additional endocrine cells: α, γ, ε, and PP cells that secrete glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide.

Using a charge-based aggregation protocol, we have developed an efficient process for forming islet-like structures from HLSCs, or HLSC-ISLets. We have shown that these structures:

  • Express both β-cell transcription factors (Nkx6.1, Nkx6.3, MafB) and endocrine specific markers (chromogranin A (CgA) and Ngn3) at 14 days of Differentiation (Navarro et al. 2019).
  • Express the exocrine pancreatic marker PDX1 and human islet-specific hormones, including insulin/C-peptide, glucagon, somatostatin, and ghrelin
  • Show β-cell gene expression commitment
  • Secrete human C-peptide in response to glucose in both static and dynamic conditions
  • In vivo, rapidly reverse hyperglycaemia in diabetic SCID mice