**4. Challenges on the path of brain organoids technology**

Brain organoid technology is a little over than decade old, and it is still in its infancy. Therefore, it is no surprise that the generated brain organoids still suffer from significant discrepancies compared to the native brain. Here will be discussed some of the important issues.

**Vascularization:** The brain is one of the most metabolically active organs in the body requiring a high oxygen supply; therefore, it has one of the densest vascularization networks [91, 92]. In most of the current protocols, the brain organoids lack vascularization. Lack of organoid vascularization of such presumably active tissues worsens the viability of the cells, causes necrosis, limits the organoid size, and disturbs the tissue structure [93, 94]. The trouble with the vascularization is mostly due to the fact that the vascular epithelia originate from different germ layers than the neurons and the macroglia [95]. In a recent report, Pham et al. used a dual culture approach by separate differentiation of endothelial cells, which afterward were cocultured with the formed brain organoids [95]. However, further research is needed to determine how functional the brain organoids are and how well they recapitulate the native brain vascularization. Another proposed approach by Mansour et al. was the implantation of the organoid in the living brain in order to be provoked vascularization from the surrounding tissues, thus securing its long-term survival [96].

**Artificial maturation/aging strategies:** Currently, most of the organoids develop and differentiate with comparable speed to the one observed during the natural neurogenesis for the particular species [47]. This can be problematic in some applications. For example, if we want to produce an appropriate neural patch for an injured patient using his/her own hiPSC, then this patch generation should happen sufficiently fast in order for the patch to serve its purpose. Alternatively, if we want to study aging-related diseases such as Parkinson's. Borghese et al. used the Notch-inhibition strategy to accelerate the neuronal differentiation of ESC *in vitro* and *in vivo* [97]. However, Notch signaling plays an important role in brain patterning and neuronal specification so that such inhibition may interfere with the desired differentiation results. Miller et al. used progerin (truncated form of lamin, which is associated with premature aging) to induce aging human iPSC [98]. Later Vera et al. proposed another approach by downregulation of telomerase which induces telomere shortening [99]. Recently, Li et al. developed organoids with accelerated growth by using mutant cells; however, such genetically modified organoids cannot be used in translation [100].

**Morphological discrepancies with native brain:** One of the commonest problems with current brain organoids is that within a single organoid are formed several neuroepithelial rosettes, each acting as a separate center of morphogenesis, while in the normal embryo is formed only one. Recently Knight et al. proposed a protocol for single rosette generation [101]. They achieved a high percentage of single rosette organoids by imposing geometrical confinement on the growth in custom micropatterned plates and/or with ROCK-inhibition.

**Glial cells:** Most of the brain organoids have little or no glial cells. This is not a big surprise if we consider the gliogenesis timeline during the normal ontogenesis. Significant amounts of astrocytes start to be generated only at the late stages of the embryogenesis, while for the oligodendrocytes, this time starts with the postnatal period [102, 103]. Howbeit, the situation is very different for the microglia, which start to invade the neural tube relatively early in the embryogenesis, and by its end, all the microglia are present [103]. They are absent from the brain organoids because they are produced by different germ layers than the neuroectoderm. So, the lack of glia in the brain organoids is logical, and if we want brain organoids to be better copies of the real brains, then we either need to grow them for a comparable time length of the in vivo brain development or to find shorter ways (for the macroglia) or to introduce somehow the missing germ layer in the system (for the microglia).

There is some recent progress in the resolution of these issues. Ormel et al. modified the unguided protocol of Lancaster et al. by tweaking its timeline and reducing the concentrations of some of the additives, and as a result, they got whole-brain organoids with innate microglial cells [104]. However, this protocol may not be a feasible solution for the researchers who need more specific organoids. Later Bejoy et al. developed a different strategy by dual culturing the brain organoids and the microglia mesodermal progenitors and afterward co-culturing them so that the microglia can invade the organoids [105]. As for the macroglia, Paşca et al. and later Yakoub generated brain organoids that developed together neurons and astrocytes, howbeit they were forebrain specific [42, 106]. Recently Shaker et al. published a protocol for cortical brain organoids that could be used to develop myelinating oligodendrocytes along with astrocytes [107]. All these are encouraging results, so hopefully, we expect to see organoids with full-spectrum glial cells in the future.
