Successful transplantation of human hepatic stem cells with restricted localization to liver using hyaluronan grafts

Abstract

Cell therapies are potential alternatives to organ transplantation for liver failure or dysfunction but are compromised by inefficient engraftment, cell dispersal to ectopic sites, and emboli formation. Grafting strategies have been devised for transplantation of human hepatic stem cells (hHpSCs) embedded into a mix of soluble signals and extracellular matrix biomaterials (hyaluronans, type III collagen, laminin) found in stem cell niches. The hHpSCs maintain a stable stem cell phenotype under the graft conditions. The grafts were transplanted into the livers of immunocompromised murine hosts with and without carbon tetrachloride treatment to assess the effects of quiescent versus injured liver conditions. Grafted cells remained localized to the livers, resulting in a larger bolus of engrafted cells in the host livers under quiescent conditions and with potential for more rapid expansion under injured liver conditions. By contrast, transplantation by direct injection or via a vascular route resulted in inefficient engraftment and cell dispersal to ectopic sites. Transplantation by grafting is proposed as a preferred strategy for cell therapies for solid organs such as the liver.

Figure 1. Stability of hepatic stem cell phenotype in cells cultured in biomaterials comprised of matrix factors found in the liver’s stem cell niches

a) Gene expression in stem cell colonies cultured in Kubota’s Medium (KM) and on culture plastic versus in cells cultured three-dimensionally (3-D) in hyaluronans or in HA supplemented with type III collagen and laminin, all components known to be in the liver’s stem cell niche. Expression is normalized to GAPDH, and fold changes are normalized to those in hHpSC colonies on plastic. A significance level of p<0.05% (*) is seen that in cultures on plastic versus those in hyaluronan conditions. That comparing the expression in HA alone versus HA with collagen III and laminin are denoted by (**) b) Expression of EpCAM and NCAM in 3-D HA colonies. Higher levels of EpCAM and NCAM are seen in HA supplemented with collagen III and laminin.

Figure 2. Hepatic Functional Assays over 18 days of 3-D cultures in Kubota’s Medium and in matrix components found in human hepatic stem cell niches

Levels of albumin (a), transferrin (b), and urea (c) production in 3-D cultures in hyaluronans. The levels are normalized per cell. The cells remain stable and functional over weeks under these conditions.

Figure 3. Total flux signal captured by Luciferin-expressing, transplanted cells in grafts of cells in hyaluronans versus injected as a cell suspension in healthy and CCl4 -induced liver injury models

Flux readings are normalized to those in control animals receiving no cell transplant. Much higher flux signals are observed in grafted cells, both in the healthy and injured models, most noticeably in the first 12 and 24 hrs post-transplant. By 72 hrs, the signal is lost in all cases due to silencing mechanisms involving methylation of the CMV promoter in the adenoviral vector.

Figure 4. In vivo real time imaging of luminescent signal produced by luciferin-expressing cells in hyaluronan grafts versus injected as a cell suspension

The signals seen in animals that were grafted are found entirely in the liver, whereas those that were transplanted without grafting materials yielded a weaker, dispersed signal throughout the abdominal cavity.

Figure 5. In vivo real time imaging of luminescent signal produced by luciferin-express cells in hyaluronan grafts versus injected as a cell suspension

Shown is a replicate experiment from that demonstrated in Figure 4. The distribution of cells to ectopic sites, such as lungs, is evident in animals with direct injection. The loss of signal at 72 hrs has been shown by others (34) to be due to silencing of the CMV promoter by methylation.

Figure 6. Humanization of host livers is facilitated by grafting strategies

Grafting of transplanted cells in biomaterials such as hyaluronans, key components in the liver’s stem cell niche, results in localization of the transplanted cells to the target organ and more rapid humanization of that organ. By contrast, transplantation of cell suspensions without grafting biomaterials results in smaller numbers of human cells within the target organ and also in human cells found at ectopic sites such as lung (data not shown). Identification of the human cells is by immunohistochemistry for human albumin. Controls include livers from animals that were not transplanted and human fetal liver with and without primary antibody staining.