Learn about the key research discoveries leading to the development of modern HUB Organoids.
Milestone Discoveries in the Development of HUB Organoids
Today’s modern adult stem cell-derived HUB Organoids are the outcome of a massive research effort, spanning decades and encompassing a series of historical research milestone discoveries in stem cell culturing.
Early Milestone Discovery: Stem Cell Cultures for Developmental Biology Studies
The first ex vivo stem cell expansion studies focused on establishing long-term stem cell cultures to model human development. These efforts date back to the late 1970s and early 80s, when adult keratinocytes or embryonic pluripotent cells were co-cultured with a layer of fibroblast feeder cells. These provided a cellular source of undefined stem cell niche factors, though this was unknown at the time.
Despite these efforts, stem cells grown in the presence of feeder cells quickly became genetically unstable and lost their full differentiation potential. This means that in vitro differentiation could be somewhat limited and unpredictable. As a precautionary step, cultured (putative) stem cells were transplanted into mice to see whether they still possessed stem cell defining characteristics of: 1) the capacity to self-renew and 2) differentiation into multiple lineages.
Subsequent Milestone Discoveries: Enhanced Understanding of the Stem Cell Niche and the Regulation of Stem Cell Self-Renewal and Differentiation
Two subsequent discoveries resulted in optimized protocols for establishing organoids from human multipotent organ-specific adult stem cells (ASCs), now known as “HUB Organoids”.
The first milestone published by the Clevers lab, was the discovery of Lgr5 as a biomarker to identify adult intestinal stem cells. This discovery allowed the characterization, purification, and in vitro expansion of the first ASCs.
The observation that these ASCs proliferate and can be maintained in long term culture led to using isolated Lgr5+ stem cells as a putative cellular source for organoid cultures. This seminal work resulted in developing the first gut “mini-organs” (specifically, crypt-villus organoids).
The second major discovery was an enhanced understanding of the stem cell niche, and of the factors that regulate stem cell self-renewal and differentiation. This key aspect meant that feeder cells were no longer required. Instead, the stem cell culture environment is simply supplemented with organ-specific cocktails of growth factors.
The Clevers research group shattered the common dogma by showing that self-organizing near-native intestinal epithelial structures could be generated from single stem cells in the absence of a mesenchymal cellular niche.
Modern HUB Organoid Culture
Modern HUB Organoid culture is based on refined protocols optimized by the Clevers lab and HUB. This includes the optimization of organoid culture medium over a number of years. The refined media allows the generation of organoid cultures from epithelial ASCs in the absence of a mesenchymal cellular niche, and includes
- Stem cell niche factors: epidermal growth factor (EGF), Noggin, and R-spondins following discoveries showing that
- Wnt signaling is an essential cellular signaling pathway for stem cell maintenance in vivo.
- EGF is a potent mitogen.
- R-spondins, later identified as Wnt agonists binding to LGR5, are mitogens causing stem cell hyperplasia.
- The bone morphogenetic protein (BMP) inhibitor “Noggin” is crucial for the maintenance of the stem cell niche.
- Wnt signaling is an essential cellular signaling pathway for stem cell maintenance in vivo.
- A rho kinase (ROCK) inhibitor following work showing that adding it to primary cultures successfully inhibited anoikis (a form of cell death that occurs in anchorage-dependent cells) previously observed in purified colonic epithelial cells.
- Finally, embedding purified ASCs in Matrigel provided a key ex vivo substitute for the extracellular matrix (ECM). This was selected after previous experiences with feeder layer cultures showed that feeder cells (i.e. fibroblasts) produce ECM to support stem cell growth. Preincubation of the tissue culture plastic with ECM proteins (such as collagen or laminin) further enhanced stem cell clonogenicity. Also, ECM-based hydrogels, such as Matrigel®, could foster 3D aggregation and polarization of (stem) cells.
Using these approaches modern HUB Organoids are now derived from range of epithelial tissues.
Modern HUB Organoid Discoveries - What's Next?
Recent discoveries are allowing organoid cultures to become useful in assessing immunotherapies.
While HUB Organoids do faithfully recapitulate human epithelial organ morphogenesis and pathogenesis, they lack the communication between organs found in complex in vivo organisms. Specifically, for a disease such as cancer, co-operation between the tumor itself and the immune system is crucial for disease establishment and progression. Both cancer and immune cells are therefore needed to effectively evaluate immunotherapies.
New studies show that tumor organoids can be co-cultured with autologous activated T cells. Co-cultures of autologous tumor organoids and peripheral blood lymphocytes were used to enrich tumor-reactive T cells from the peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. These T cells were then used to assess the efficiency of killing matched tumor organoids.
Conclusion
Modern organoid cultures are the result of several historical research discoveries, including early studies showing stem cells could be cultured in vitro, followed by later studies that improved our understanding of the stem cell niche and the regulation of stem cell self-renewal and differentiation.
Today, optimized HUB Organoid protocols are widely used to generate robust and reproducible organoid cultures that can be maintained long-term in vitro.
