Introduction

Due to increased industrialization in modern era, the chances of exposure to different known/unknown chemicals have been increased exponentially. Adults, children and developing foetus are routinely being exposed to variety of environmental chemicals including pollutants, drugs and new chemical entities (NCEs). Exposed people may not have any detrimental effect during short-term period, but it could have serious consequences during long-term exposure because of different systemic toxicities including neurotoxicity. The developing brain of child as well as of foetus has been found much more vulnerable towards the exposure of different environmental xenobiotics including organophosphate pesticides [1, 2]. The developing brain of foetus/newborn is always at higher risk against the exposure of environmental chemicals because of underdeveloped placental barrier as well as blood-brain barrier [3–5]. The high lipid contents and lower regeneration/post-mitotic nature of neurons work as oil in the fire and may enhance oxidative stress-mediated cell death after the exposures of environmental contaminants. The brain development is a very complex process which involves several other processes running in parallel such as cell proliferation, migration, homing, differentiation and synapse formation in highly regulated manner [6, 7]. Hampering any step at any stage of these cellular processes due to the exposure of xenobiotics/drugs/NCEs could lead to the life-long permanent disability [8–10]. Plethora of literature is available on public domain showing the susceptibility of developing human brain towards many toxicants/ chemicals and further development of neurological deficits/disorders [6–8]. Thus, the developing brain is much more critical towards the exposure of environmental chemicals in respect to developed adult brain which may not have severe consequences. Many studies of developmental neurotoxicity (DNT) involved traditional in vivo models using large number of experimental animals. Due to rapid industrialization, numbers of new chemicals are exponentially increasing in our ecosystem which needs to be tested precisely in term of developmental neurotoxicity in respect of animal welfare. For the assessment of developmental neurotoxicity of these environmental chemicals, in vivo animal models are not suitable as animal testing is complex, time consuming, costly and requires considerably high numbers of laboratory animals. To address the issue, 3Rs (reduce, refine and replace) concept has been adopted to develop alternative in vitro models which could reduce and refine the animal usage for rapid DNT evaluation. Moreover, the data generated through such in vivo animal studies are very difficult to recapitulate and extrapolate to human beings. Furthermore, the non-availability of human fetal brain tissue due to strict ethical problem makes this field very difficult and challenging. Therefore, to address these issues, many in vitro models derived from brain cells have been used for the assessment of neurotoxicity/developmental neurotoxicity which have shown less ethical dubious and provided more predictive and sensitive tool for functional studies at both cellular and molecular levels [9–11]. The major advantage of these cell-based in vitro models is their ability to reproduce various complex stages of brain development at cellular and molecular level. The literature is full of reports showing the use of these types of in vitro models for neurotoxicity/DNT studies involving different neuronal cell lines namely, rat pheochromocytoma PC12 cells [12–14], human neuroblastoma-SH-SY5Y cells [15–18], primary cultured neuronal/glial cells [19, 20], rat cerebellar granule cells [21–24] and cortical neurons [25, 26] to understand cellular and molecular mechanism of DNTs [27–31]. The established cell lines propagate rapidly and provide a homogenous population of cells which can be differentiated into neuronal-like cells by using various growth factors/neurotrophins. The only concern remains here is that these in vitro models utilizing cell lines which are genetically instable and terminally differentiated cells. Because of genetic instability, these cell line-based in vitro models may have different physiological outcomes after the exposure to toxicants in comparison to in vivo situations, and terminally differentiated nature ceases them to mimic the accurate evolutionary differentiation process. Thus, it is very difficult to extrapolate data generated through such in vitro models with the data generated by animals under in vivo situations. On the other side, the use of primary cultures of human neuronal/glial cells is hampered because of non-availability of developing/mature human brain tissues. In addition, primary cultures also contain post-mitotic neurons and have relatively limited lifespan [11, 32].

Stem cells are known to have self-renewal capability, long-term proliferation and plasticity potential towards the development of variety of cell types including brain neuronal and glial cells. Therefore, stem cells from different sources are particularly suited to study DNTs [32–36]. In the last few decades, establishment of stem cell-based in vitro model systems for DNT assessment has been the subject of high thrust in all over the world. Thus, we can say that stem cells are better promising and unparalleled tools for developing unique in vitro model systems to study developmental neurotoxicity. Theoretically, data generated from these in vitro model systems will be free from different concerns raised because of the genetic instability and terminal differentiation. Furthermore, it is easy to extrapolate the data generated employing human stem cells to predict/anticipate DNTs in human beings. Various types of stem cells based on their sources viz., embryonic stem cells [37–40], neural stem cells [41–43], bone marrow stem cells [44, 45] and umbilical cord blood stem cells [33–36, 43, 46–49] are being explored to develop alternative in vitro models for developmental neurotoxicity (DNT). The present review summarizes about the latest advancements/progresses achieved in the development and validation of human umbilical cord blood stem cells based in vitro models to evaluate the developmental neurotoxicity and their possible application in therapeutic pre-screening of various environmental chemicals, toxicants, pesticides and drugs.

Umbilical Cord Blood has Diverse Population of Stem Cells

Human umbilical cord blood (hUCB) is a perfect and one of the thriving source of haematopoietic stem cells (HSCs), non-haematopoietic stem cells (non-HSCs) and progenitor cells [48–56] and does not have any ethical concern as placenta is generally discarded after the birth of child. Therefore, the use of cord blood stem cells is non-controversial, very cheap and the most suitable biological material for DNT study. The non-invasive collection makes it comparatively cheaper and wonderful tool for establishing in vitro models of DNT [57]. Furthermore, it is very easy to grow these hUCB-HSCs, non-HSCs, mesenchymal stem cells (MSCs) and multipotent progenitor cells under in vitro conditions in undifferentiated state without any major loss in the pluripotency potential. In order to determine optimal conditions for in vitro expansion of human umbilical cord blood stem cells (hUCBSCs), researchers have tried various types of culture medium along with different permutation combinations of various cytokines, growth factors and physical parameters [58–61]. Various growth factors such as basic fibroblast growth factor (bFGF), stem cells factor (SCF), thrombopoietin (TPO), Fms-like tyrosine kinase-3 ligand (Flt-3 ligand), insulin-like growth factors (IGF-1&2) and cytokines (IL-3, IL-6, G-CSF, GM-CSF) have been found to be promising factors for efficient expansion of hUCBSCs specifically HSCs. These growth factors and cytokines are known to play key role in early haematopoiesis and prolonged undifferentiated proliferation of stem cells. TPO and Flt-3 ligand are known to be critical to regulate early proliferation and suppress apoptosis and ageing in hUCB-HSCs as well as in progenitor cells during in vitro culture conditions. Stem cell factor interacts with specific c-kit receptor and triggers signalling cascade to promote haematopoiesis and stemness and also maintains suitable microenvironment of haematopoietic stem cells and progenitor cells. Basic fibroblast growth factor (bFGF) significantly reduces cellular senescence and promotes stem cell self-renewal and differentiation [58, 62–68]. The protocols for isolation and long-term proliferation of hUCBSCs have been well established now.

Human umbilical cord blood-derived HSCs/non-HSCs as well as progenitor cells have high commitment towards different specialized lineages including brain cells of ectodermal origin. Buzanska and colleagues did pioneer work in this area and successfully isolated, characterized and differentiated these hUCBSCs into neuronal cells [46]. We and others have also successfully purified primitive HSCs (CD34+/CD133+/Thy1+) and CD34−/CD133− non-HSCs from whole human umbilical cord blood [34–36, 46, 48, 49]. We have reported the prolonged maintenance and substantial expansion of these human cord blood-derived CD34+/Thy1+ primitive HSCs, which also having extensive self-renewal, long term proliferation capacity and clonogenic capabilities. hUCBSCs express pluripotency markers such as Oct-3/4, Sox-2, Nanog and c-Myc which are usually expressed in pluripotent embryonic stem cells and thought to play key roles in maintaining pluripotency and self-renewal capabilities [49, 69–71]. Similarly, we also found high expression of these pluripotency markers namely, Oct-3/4, Sox-2, Nanog and c-Myc in CD34+/Thy1+ hUCB-HSCs, and moreover, the expression levels of these pluripotency markers were reduced during the differentiation of these hUCBSCs into neuronal cells [34–36]. These purified and characterized populations of hUCBSCs have great plasticity potential towards various specialized cell types of all three germ layers [72]. Although hUCB-HSCs are categorized as pluripotent stem cells, various other types of non-stem cell population are also present in the whole cord blood and must purify before carrying out to achieve quality differentiation for error free DNT studies. Additionally, human umbilical cord blood has been well accounted to contain a rich population of mesenchymal stem cells (MSCs) expressing many specific cell surface markers namely, CD29, CD44, CD90, CD105 and CD273 [73–76]. The connective tissue layer of human umbilical cord, Wharton’s jelly, is also a copious source of MSCs [77, 78]. The plasticity potential of MSCs is incomparable with umbilical cord blood stem cells as MSCs cannot be differentiated into various cell types of all three germ layers [79], but these cells have high potential to differentiate into neuronal cells under the influence of various neurogenic growth factors/neurotrophins [79–86]. Pluripotency of hUCBSCs could also have been enhanced by transfecting these cells with pluripotency-associated transcription factor genes namely, Sox-2, Oct-4, Klf-4 and c-Myc [87, 88]. Buzanska and colleagues successfully established hUCB-derived neural stem cell (NSC) line having the ability of higher growth, self-renewal capacity and plasticity potential towards neural cells [33, 46]. Our group has also demonstrated that Wnt/GSK3β/β-catenin signalling play a crucial role in the normal proliferation and maintenance of hUCBSCs, and pesticide-challenged cells rapidly enter into apoptosis [89]. Thus, easier accessibility of placental tissue/blood, diverse proliferating population and enormous plasticity makes these haematopoietic stem cells a very powerful tool to study developmental neurotoxicity of various xenobiotics, toxicants, pesticides and NCEs.

Human Umbilical Cord Blood Stem Cells Easily Differentiates into Neuronal Cells

Although embryonic stem cells have the maximum pluripotency power as well as plasticity potential, but they also have their own limitations and have been avoided for transplantation purposes or for other DNT studies. The main problems associated with the use of embryonic stem cells are ethical, religious and political as they represent a complete embryo. Additionally, unrestricted cell growth of these embryonic stem cells could lead to the formation of teratomas even after the differentiation into specific cell types [90, 91]. Neural stem cells derived from specific brain region of human may be the best source for developmental neurotoxicity as these cells do not have intergenomic epigenetic variations due to similar genetic material; however, this application is limited due to ethical problem and the least regenerative power of brain tissue [92]. These limitations are major hurdles to use human embryonic or neural stem cells for creating novel alternative in vitro model for DNT studies. Alternatively, hUCBSCs could serve the purpose and have been proved to be the most promising in vitro tools for DNT studies [33–36, 47]. There is no concern about teratoma formation in the neuronal cells derived from hUBSCs and also have almost no ethical concern. Moreover, these cells are considered as one of the most enriched source of stem cells [48, 49, 53]. The non-invasive collection method makes it excellent tools to study developmental neurotoxicity [34–36, 48, 54, 57].

Pluripotent stem cells derived from human umbilical cord blood have similar potential of neuronal differentiation as neural stem cells derived from foetus [34–36, 93–99]. Neurons derived from these hUCBSCs have expression of different early and mature neuronal markers namely, nestin, musashi-1, nectin, neuronal nuclei (NeuN), post-synaptic density protein 95 (PSD95), synaptophysin (SYP), β-III tubulin (TUJ-1), growth-associated protein 43 (GAP43), various forms of neurofilaments (NF), neurotrophic growth factors and neuron-specific receptors N-methyl-d-aspartate (NMDA) and γ-aminobutyric acid (GABA) [34–36, 38, 53, 86, 94, 100–107]. Our group have also successfully isolated, maintained and differentiated hUCB-derived CD34+ HSCs into neuronal cells using nerve growth factor (NGF) and trans retinoic acid in serum free neurobasal medium. These differentiated cells displayed a typical neuron-like morphology and expressed significantly various early-stage, mid-stage and mature neuronal markers namely, nestin, synaptophysin, neuronal nuclei, PSD95, NFM, NFH, TUJ-1, MAP2, GAP43, PSA-NCAM, acetyl cholinesterase, neuron-specific receptors such as AMPA receptor, NMDA receptor (NR2A), neurogenesis transcription factor CREB and neuron-associated growth factors such as NGF and BDNF, etc. (Table 1). Moreover, we observed the decreasing expression of stem cell and pluripotency markers namely, CD34, CD133, c-MYC, OCT3, SOX2, Nanog and SHH throughout the neuronal differentiation [34–36].

Table 1 List of marker genes (stem cell markers, neuronal markers, markers of xenobiotic metabolism, apoptosis, oxidative stress and various mitogen-activated protein kinases) selected to study the mechanism of chemical-induced developmental neurotoxicity in differentiating neurons derived from human umbilical cord blood stem cells [34–36, 89]
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In addition, hUCB-MSCs have also been extensively studied for neurogenic potentials [105, 81, 108–111]. Recently, Zhang and colleagues [76] used condition medium-constituted olfactory ensheathing cells for the differentiation of mesenchymal stem cells into neuronal cells. These neuron-like cells were positive for neuron-specific enolase and having similar neuronal electrophysiological properties. Other groups have also demonstrated similar electrophysiological properties in differentiated neurons derived from hUCB pluripotent stem cells and MSCs [103, 112]. More specifically, hUCB-derived MSCs and other multipotent stem cells can also be differentiated into more specific neuronal cells like dopaminergic neurons which have higher expression of specific markers namely, DAT, TH, Nurr1, Pitx3 and dopamine transporter proteins [84, 85]. These types of cells can serve as an alternative regenerative medicine against Parkinson’s disease. Similar kind of study on hUCB non-haematopoietic multipotent stem cells showed differentiation of these cells into cortical GABAergic neurons with upregulated expression of GABAergic regulatory enzymes and transcription factors namely, MASH1 and DLX1 & 2 [86]. Even unspecified mononuclear cells derived from hUCB have capability to express neuronal markers namely, Musashi-1 and TUJ-1 and GFAP under the influence of specific growth factors/neurotrophins [113]. Seo and Cho have observed that differentiation of MSCs into neuronal cells induced the secretion of numerous trophic factors which can modulate different cellular processes such as neurogenesis, inflammation, angiogenesis and apoptosis [114].

Neurotrophins play an important role in the neuronal differentiation of hUCBSCs. Neurotrophin and NGF play very critical roles in the survival, maintenance and differentiation of sympathetic and sensory neuronal pathways. Binding of NGF to transmembrane tyrosine kinase receptor (TrkA) facilitates receptor dimerization and phosphorylation at cytoplasmic site which further facilitates phosphorylation of cytoplasmic adaptor protein (Shc) and initiates cell survival through AKT/MAPK pathway which suppresses c-JUN through CREB. NGF plays an important role in maintaining body haemostasis as binding of NGF/pro-NGF with lower affinity to p75NTR receptor could also lead to either survival through NF-kB or cell death through c-Jun N-terminal kinase activation [115]. It is well known that BDNF induces phosphorylation of MAPK/ERK and β-catenin through tropomyosin receptor kinase B (TrkB receptor) and triggers PI3K/AKT-dependent signalling pathways to stimulate neural differentiation and cell survival of hUCB-HSCs and MSC-derived neuronal cells [81, 89, 108]. We and others have described the importance of neurotrophins in the neuronal differentiation from hUCBSCs [34, 36]. Several studies showed that neurotrophins are induced endogenously during neuronal differentiation and make this process more viable [34, 36, 116]. Hafizi and colleagues have demonstrated the role of neuro miRNAs (mir-9 and mir-124) which played critical roles in differentiation of neuronal cells from CD133+/CD34+ hUCB-HSCs. Micro RNAs (miRNAs) could also play an important role in hUCBSC-derived in vitro DNT models and may precisely describe mechanism of developmental neurogenesis process [99]. Overall, the protocols are well established for the differentiation of stem cells into neural cells and more specific brain cells such as glial cells [117], cortical GABAergic neurons [86] and dopaminergic neurons [84, 85]. Thus, the morphological and physiological differentiation of hUCBSCs into neuronal cells in time-specific manner could serve as suitable in vitro model system for DNT studies without involving other issues related to ethics, cost and time.

Use of hUCBSc in the Development of In Vitro Model for Developmental Neurotoxicity

To take all the benefits into account, extensive efforts are being made worldwide to develop in vitro models for DNT studies by using hUCBSCs. Although, initially, these approaches seem very costly in terms of isolation, purification and maintenance of hUCBSCs, but once established, then we can work on relatively cheaper alternatives like replacement of neurotrophins with conditioned medium from specific neuronal cells [85, 118]. We and others have successfully proved that umbilical cord blood stem cells worked as in vitro tool to study DNTs of different chemicals/pesticides, and our studies also revealed neuronal cells derived from hUCBSCs have almost parallel expression of neuronal markers as reported during neurogenesis in foetus. We were able to differentiate hUCBSCs into neuronal cells in time-dependent manner and also characterized different stages of maturity namely, day 2, day 4 and day 8 by high throughout TaqMan low-density array-based real-time quantitative PCR and western blotting [34, 36]. We validated our differentiation process by observing decrease in the levels of different pluripotency markers namely, CD133, MYC, NANOG, SHH, KLF4, SOX2, POU5F1 and FRAP1as well as upregulation in the levels of different neuronal markers namely, NGF, BDNF, NFM, MBP, NFH, NCAM, STAT4, CHRM2 and NR42A during the progression of differentiation process (Table 1). An early increased and later downregulated level of early differentiation marker protein nestin further confirmed our differentiation process [34, 36]. Results were clearly indicating that hUCBSCs successfully differentiated into neuronal cells which could serve as novel in vitro model for developmental neurotoxicity studies. We divided differentiation process into four stages, undifferentiated, early differentiated, mid-differentiated and fully differentiated stages. We exposed these differentially differentiated cells with subtoxic doses of organophosphate pesticide monocrotophos (MCP) for a very short time, 3 h for transcriptional and 6 h for translational changes. We rule out the results of undifferentiated hUCBSCs in DNT studies because of their non-neuronal and different origin. We found higher damage in early and mid-differentiated neuronal cells with respect to fully mature neuronal cells [36]. Instead of us, several other groups also worked in this direction of developmental neurotoxicity and established the role of hUCBSCs for the assessment of DNTs of various environmental chemical entities [33–36, 43, 46, 47, 49, 102]. The researchers are also trying to establish 3D cultures of nervous system by using these hUCBSCs which seem to be the most promising and realistic in vitro model to study developmental neurotoxicity in human [33, 46, 119]. There is high probability of in vivo mimicking for developmental neurotoxicity of chemicals in in vitro 3D conditions compared to in vitro 2D conditions. Even hUCB-NSCs which were grown in bioengineered surface may have better comparable results to human being than in vivo animal data due to greater cell to cell interactions, controlled geometry and spatial distribution of the cells on the surface. This type of in vitro model of differentiating cells has been validated by exposing cells to known neurotoxicant MeHgCl [119]. Differentiated neuronal cells derived from hUCB-NSC line have been used for robust neurotoxicity assessment of a broad range of neurotoxic compounds of different categories [33]. Our data from hUCBSC-based in vitro model systems showed that MCP, a known organophosphorus pesticide, significantly altered neuron-specific MAPKs, oxidative stress, metabolism, apoptosis, neuronal and stem cell markers in early and mid-differentiation neurons. These differentiated and well-characterized neuronal cells were showing depleted dopaminergic and cholinergic receptors after the exposure of MCP, a known developmental neurotoxicant [36]. Additionally, our group did pioneer work on complete profiling of xenobiotic-metabolizing cytochrome P450s to establish developmental stage-specific bio-markers of exposure and effects using hUCB-CD34+ cell-derived differentiating neuronal cells. We have also reported that human HSC-derived developing neuronal cells expressed xenobiotic-metabolizing cytochrome P450s (CYP1A1, 2B6, 2E1 and 3A4), related receptor regulators (AHR, CAR and PXR) and phase II metabolizing enzyme GSTP1-1 during all the maturity periods. Furthermore, we also characterized responsiveness/functionalization of these CYPs using known inducers and inhibitors of CYPs along with neurotoxicant MCP [34]. Overall, results were not different from our other study that concluded early-stage differentiating neurons were more vulnerable towards toxicant compared to fully mature neurons. Thus, the HSC-derived developing neurons could be a homogenous in vitro tool to predict human-specific developmental neurotoxicity against various environmental chemicals and drugs (Fig. 1) [34, 36]. Recently, we explored the effect of 3-methylcholanthrene (MC), polycyclic aromatic hydrocarbon, on hUCB-HSC-derived developing neurons and reported stage-specific molecular mechanism of developmental neurotoxicity. Our findings suggest that MC significantly induces the expression and activity of AHR, CYP1A1 and GSTP1-1 and reduces the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-d-aspartate (NMDA) receptors as well as cAMP response element binding protein (CREB). Additionally, MC hinders phosphorylation of neurogenesis transcription factor CREB through activating AHR and interferes with neuronal transmission which could lead to impaired neurogenesis/brain functions during brain development in neonates [35].

Fig. 1
figure 1

A general approach to show the use of human umbilical cord blood stem cell (hUCBSC)-derived neuronal cell-based in vitro model to study developmental neurotoxicity The applicability of hUCBSC-derived differentiating neuronal cell-based in vitro model to assess the developmental neurotoxicity of chemicals/drugs/xenobiotics/NCEs is unparalleled. Umbilical cord blood could be used as an enriched source for the isolation of pluripotent haematopoietic stem cells. During their neuronal differentiating, these cells may be exposed to unknown chemicals/drugs/xenobiotics/NCEs, and the effects of these compounds can be assessed by studying different markers involved in cell proliferation, neuronal differentiation, neuronal injuries and receptors at various stages of neuronal maturity such as days 2, 4 and 8. These neuronal cells derived from human umbilical cord stem cells can be used as a powerful tool to assess the developmental neurotoxicity in human beings

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Conclusion

The Health Effect Test Guidelines OPPTS 8706300US by US Environmental Protection Agency (EPA, USA) and New OECD DNT Test Guidelines 426 (OECD, 2007) by Organization for Economic Cooperation and Developments mainly recommend animal use for developmental neurotoxicity studies in standard adult and developing animals. However, there is huge pressure on industries, education centres and regulatory bodies to develop methods for efficient screening of large number of chemicals/xenobiotics which may have danger of ‘silent pandemic’ or unknown adverse effects on developing brain of children. Experts from all over the world are giving more emphasis to develop in vitro models for DNT studies due to efficient screening by high throughput nature, lower cost, less assessment time and higher reliability. The preliminary screening from these in vitro models could refine animal number for such type of developmental neurotoxicity studies. First of all, there is need to define/develop new models/methods which could be used to explore the effect of different drugs/chemicals on developing nervous system afterword these alternative models/methods can be further recruited according to regulatory guidelines and requirements. Even before their regulatory acceptance, these models can prioritize different chemicals for in vivo developmental neurotoxicity assessment. We have to cover a long way before finding a suitable in vitro model who fulfills the entire requirements for integrating and interpreting developmental neurotoxicity data with respect to human beings. There should be a general understanding between regulatory agencies and scientists to know about limitations of each other in the development of these high throughputs in vitro model systems. These models should mimic the evolutionary conserved neurodevelopmental processes which make them mechanistically more relevant to human developmental neurotoxicity. Furthermore, these models should be able to decode the mechanism based on altering the cell to cell/organ to organ interactions and should be free from any ethical concern. To fulfil these requirements of least genomic variations, unavailability of human tissue and less ethical concern, neuronal cells derived from human umbilical cord blood seem to work as a powerful tool for the development of high throughput in vitro model to study developmental neurotoxicity. Human umbilical cord blood is imperishable and affluent source of haematopoietic, non-haematopoietic and progenitor stem cells. High preserving cost to preserve human umbilical cord makes it invaluable to ordinary peoples but works as positive thrust for research/industrial applications. The non-invasive collection methods, easy proliferation and high plasticity make it perfect to use for developmental toxicity studies including developmental neurotoxicity. Above all, hUCBSCs could be used for drugs/xenobiotics screening based on in vitro models of homologous cells as well as autologous cells which spur interest in designing a feedback validation process. Human umbilical cord blood derived haematopoietic, non-haematopoietic and mesenchymal stem cells as well as progenitor cells have the capability of self-renewal, long-term proliferation and easy differentiation into specific cells, and these properties make hUCBSCs as gold standard tool for establishing in vitro models of DNT. The differentiation of hUCBSCs is not only limited to nervous system but these cells can also give rise to many other cell types of different organs such as heart, kidney, retina, gut, bone, etc. and opens the new door to work on developmental toxicity for these organ too. Therefore, human umbilical cord blood stem cells have great potential to work as a fundamental tool for developing unique in vitro model system of developmental system toxicity including neurotoxicity to test broad spectrum of drugs/chemicals which almost seems impossible using animal-based in vivo model system. Models based on 3D growth of neuronal cells derived from umbilical cord blood stem cells have potential to decode the cell to cell/organ to organ interaction-based mechanism for developmental neurotoxicity. Initially, these researches may seem very costly, but it will become very cost-effective and useful after the development of suitable high throughput in vitro model(s) for developmental neurotoxicity.