Fig 1.
Cyclic development of Plasmodium species in three distinct niches of hosts.
In mammalian hosts, asexual development of all Plasmodium species begins in the liver. (A) Exo-erythrocytic (liver-stage) development: Following a blood meal, sporozoites are released from the salivary glands of a female Anopheles mosquito into the skin. They travel through blood vessels and invade hepatocytes. Inside the hepatocytes, sporozoites undergo multiple rounds of cell division—schizogony—to produce schizonts. In the case of P. vivax and P. ovale, some sporozoites enter a dormant, non-proliferative stage called a hypnozoite. Once schizonts fully mature, merosomes egress from the infected hepatocytes into the bloodstream. (B) Erythrocyte-stage development: Merozoites invade red blood cells and undergo schizogony to generate additional merozoites. Some ring trophozoite stages differentiate into male and female gametocytes, which are transmitted to a female Anopheles mosquito during a subsequent blood meal. (C) Sporogony-stage development: In the mosquito midgut, ingested gametocytes fertilize to form zygotes. These develop into ookinetes and then oocysts, which produce numerous sporozoites. The sporozoites migrate to the mosquito’s salivary glands and await transmission to a human host at the next blood meal. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/qbcziwc.
Fig 2.
Two-dimensional (2D) cell culture systems for modeling liver-stage malaria.
To study liver-stage development of Plasmodium spp., several human hepatic cell types are used: primary human hepatocytes (PHHs), hepatocellular carcinoma lines (e.g., HepG2-A16 and HHS-102), immortalized hepatic cell lines (e.g., HC-04), and induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells. Hepatocellular carcinoma and immortalized cells typically grow as monolayers on rigid plastic surfaces. In contrast, PHHs must be cultured on collagen type IV-coated surfaces or co-cultured with murine fibroblasts. Sporozoites are introduced into the culture well, often followed by centrifugation or reduced medium volume to enhance sporozoite-cell contact. Successful liver-stage development is commonly indicated by segmented schizonts, which serve as a proxy for parasite development. Detection of free merozoites in the culture medium is less frequent in most available liver-stage assays. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/3mgz0tq.
Fig 3.
Liver development and differentiation of pluripotent stem cells (PSCs) into hepatocytes.
Insights into liver development largely arise from mouse embryonic studies. Applying these principles to human PSCs, such as embryonic stem cells or iPSCs, reveals that external stimuli (e.g., growth factors, hormones) drive PSCs to differentiate into hepatocytes. (A) External stimuli in hepatocyte generation from PSCs: PSCs are first induced to form definitive endoderm (DE) using signals from Nodal, bone morphogenic protein (BMP), fibroblast growth factor-a (FGFa), and Wnt pathways. Once committed, DE differentiates into hepatic endoderm (HE), which can give rise to pancreatic cells (P), cholangiocytes (Ch), and hepatocytes (H). Distinct sets of stimuli direct HE to differentiate into either hepatocytes or cholangiocytes. (B) Mouse liver budding: In mouse embryos, the foregut endoderm, derived from DE, forms the liver, pancreas, and gall bladder. The hepatic endoderm (HE) migrates into the septum transversum mesenchyme (STM), where it differentiates into hepatoblasts and then hepatocytes. Hepatic and endothelial cell formation in STM creates a bud-like structure, resembling a developing liver bud. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/nrtq41n.
Fig 4.
Methods for generation of liver organoids.
Various methods produce liver organoids, depending on the cell source and 3D structure formation strategy. Currently, liver organoids can be generated from primary human hepatocytes (PHHs) or PSCs. PSC-derived hepatic endoderm (HE) or hepatocytes (H) can form 3D organoids through stages involving definitive endoderm (DE), HE, hepatoblast (Hb), and eventually H. PHHs may originate from fetal or adult livers. To create a 3D structure, cells are embedded in a gel-like scaffold, such as Matrigel (an extracellular matrix-based medium), then covered with liquid medium. Following the principles of mouse liver budding, combining HE, endothelial cells (EC), and STM on top of solidified Matrigel can produce a liver bud-like structure. Created in BioRender. Kulkeaw, K. (2025) https://BioRender.com/kkfupxg.