**2. Organoids origin, structures and culture**

Organoids can be derived from either [1] pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) or [2] multipotent organ-specific adult stem cells (AdSCs). Both approaches take advantage of the endless expansion potential of stem cells in culture. Also, when PSCs and AdSCs are allowed to differentiate in culture, they display a remarkable capacity to self-organize into structures that reflect similar characteristics of the organ they attempt to mimic [7].

PSCs can be differentiated into different cell types and grown *ex vivo* as organoid models by the treatment with defined developmental stimuli. PSCs isolated both from mouse and human tissues have given rise to brain, retina, inner ear, stomach, intestine, thyroid, lung, liver, and kidney organoids. ESCs or iPSCs can be derived *in vitro* into endoderm, mesoderm and ectoderm, with specific procedures involving multiple differentiation steps. Thus, human iPSCs are sequentially exposed to a progression of differentiation signals in order to simulate the stages of a human developmental process. Once the initial germ layer has arranged, cells are transferred into 3D systems [8], where differentiated iPSCs aggregate to form an organ bud and, later on, organoids. These organoids contain multiple cell types and faithfully mimic the mature organ structure, and the interactions between them.

As an example, embryoid bodies (EBs), 3D aggregates of PSCs, originate cerebral organoids and develop into a forebrain region in the presence of growth factors (i.e., hFGF basic, ROCK inhibitor, N2, Heparin, MEM-NEAA, etc.). For other organs, the addition of Activin A to PSCs specifies them towards an endodermal fate. These cells are further cultured as 3D organoids in Matrigel with medium containing tissue-specific growth factors [9].

On the other hand, AdSCs-organoids can be originated from isolated adult stem/progenitor cells or from isolated tissue fragments of the corresponding organ (e.g. intestinal crypts, liver or pancreas ducts) [8]. These structures can be generated from biopsies isolated directly from the organ of interest or from diseased patient tissue without the complicated process of reprogramming and differentiation required in iPSC organoids. In general, human AdSCs-derived organoids are composed mainly of cell types found in the epithelium.

AdSCs were long believed to be unable to proliferate outside the body, but the culture with specific growth factor cocktails mimicking stem cell niches, has helped to sort out such obstacle. These niche factors are essential to support stem cell

*Organoids Models for the Study of Cell-Cell Interactions DOI: http://dx.doi.org/10.5772/intechopen.94562*

activity and vary depending on the tissue of origin. Also, 3D Matrigel-based cultures have provided the appropriate culture conditions to generate AdSCs-derived organoids from various mouse and human tissues including the colon, stomach, liver, lung, prostate, pancreas, ovaries, taste buds, and lingual epithelium.

Thus, to generate AdSCs-organoids a tissue biopsy is cut into fine particles and then incubated with enzymes (i.e., collagenase, elastase, or dispase) to obtain a single cell suspension. Next, cells are grown in Matrigel and culture medium supplemented with specific tissue growth factors [9]. For example, intestinal organoids need Noggin, R-spondin, Epidermal growth factor (EGF), and WNT [10–12]; retina organoids need IWR1e and Smoothened agonist CHIR99021 [13, 14]; prostate organoids require Noggin, R-spondin and EGF [15], while pancreas organoids require Noggin, R-spondin, EGF, fibroblast growth factor (FGF) and Nicotinamine [16].

AdSCs organoids do not require genetic transduction with transcription factors, as it happens with those with PSCs. This situation makes organoids physiologically well-suited with the host tissue, leading to an improved stem cell transplantation. Moreover, molecular techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (CRISPR-Cas9) genome technology and single-cell RNA sequencing, can be applied to organoids [7, 9]. On the other hand, the establishment of human AdSC-derived organoids is limited by the accessibility to the tissue and prior knowledge of the culture conditions for that tissue. However, an iPSC line, once established from a patient, can generate different tissue models without any time limit, beyond the patient's lifespan [17, 18].
