Fetal Neural Cells Research Articles

Cryopreservation of rat fetal neural cells as a component of suspension and aggregates

Cryopreservation of rat fetal neural cells as a component of suspension and aggregates

The miRNA transcriptome of cerebrospinal fluid in preterm infants reveals the signaling pathways that promote reactive gliosis following cerebral hemorrhage.

Germinal Matrix-Intraventricular Haemorrhage (GM-IVH) is one of the most common neurological complications in preterm infants, which can lead to accumulation of cerebrospinal fluid (CSF) and is a major cause of severe neurodevelopmental impairment in preterm infants. However, the pathophysiological mechanisms triggered by GM-IVH are poorly understood. Analyzing the CSF that accumulates following IVH may allow the molecular signaling and intracellular communication that contributes to pathogenesis to be elucidated. Growing evidence suggests that miRs, due to their key role in gene expression, have a significant utility as new therapeutics and biomarkers. The levels of 2,083 microRNAs (miRs) in 15 CSF samples from 10 infants with IVH were measured using miRNA whole transcriptome sequencing. Gene ontology (GO) and miR family analysis were used to uncover dysregulated signalling which were then validated in vitro in human foetal neural progenitor cells treated with IVH-CSF. Five hundred eighty-seven miRs were differentially expressed in the CSF extracted at least 2 months after injury, compared to CSF extracted within the first month of injury. GO uncovered key pathways targeted by differentially expressed miRs including the MAPK cascade and the JAK/STAT pathway. Astrogliosis is known to occur in preterm infants, and we hypothesized that this could be due to abnormal CSF-miR signaling resulting in dysregulation of the JAK/STAT pathway - a key controller of astrocyte differentiation. We then confirmed that treatment with IVH-CSF promotes astrocyte differentiation from human fetal NPCs and that this effect could be prevented by JAK/STAT inhibition. Taken together, our results provide novel insights into the CSF/NPCs crosstalk following perinatal brain injury and reveal novel targets to improve neurodevelopmental outcomes in preterm infants.

Fetal neural precursor cell treatment for PD.

Fetal neural precursor cell treatment for PD.

First Clinical Report on the Treatment of Parkinson's Disease with Fetal Midbrain Precursor Cells.

Because human fetal ventral mesencephalic tissue grafts provide promising results in ameliorating Parkinson's disease-implicated motor dysfunctions, human fetal midbrain-derived dopamine neuronal precursor cells are considered good candidates for cell-based therapy for Parkinson's disease in that large quantities of cells can be supplied through a good manufacturing practice-compliant system. We conducted a prospective, phase I/IIa, dose-escalation, open-label "first-in-human" clinical trial with fetal neural precursor cells to assess their safety and therapeutic efficacy in patients with idiopathic Parkinson's disease. Fifteen patients were assigned to receive three different doses of cells (4 × 106 , 12 × 106 , and 40 × 106 cells) and completed a 12-month follow-up. The primary outcome was safety, by measuring the presence of grade 3 or higher cells according to National Cancer Institute guidelines and any contaminated cells. Secondary outcomes assessed motor and neurocognitive function, as well as the level of dopamine transporters, by positron emission tomography-computed tomography. Although a pronation-supination and hand/arm movement performance was remarkably enhanced in all three groups (all P < 0.05), the medium- and high-dose-treated groups exhibited significant improvement in Unified Parkinson's Disease Rating Scale Part III only up to 26.16% and 40%, respectively, at 12 months after transplantation without any serious clinical complications or graft-induced dyskinesia in all patients. However, the motor improvements did not correlate with increase in the dopamine transporter on positron emission tomography images. Our results primarily demonstrate the safety and plausible dose-dependent efficacy of human fetal midbrain-derived dopamine neuronal precursor cells for idiopathic Parkinson's disease. © 2023 International Parkinson and Movement Disorder Society.

Characteristic of migration in culture of newborn and fetal rat neural cells

Characteristic of migration in culture of newborn and fetal rat neural cells

Current Knowledge and Future Directions in Huntington’s Disease

Introduction: Huntington's Disease (HD) is a hereditary, neurodegenerative disorder due to abnormal repeats of the CAG triplet in the IT-15 gene. It is characterized by a triad of progressive motor, psychiatric and cognitive symptoms, resulting from striatal neuronal loss. The impact of HD in Latin America is evidenced by the prevalence rates in Maracaibo (Venezuela) and Juan de Acosta (Colombia), which are the highest recorded in the world. This contrasts with the social abandonment and the scarce intervention of local governments. Aim: Provide an approach to the most relevant aspects of HD from its pathogenesis and associated genetic polymorphisms, to the current treatment options. Methodology: A literature review was performed of the state of the art of prognosis and treatment strategies in HD, including the identification of different polymorphic markers in the genes coding for UCHL1, HIP1, PGC1α, GRIK2, TBP, BDNF, among others, and its associations in the evolution of the disease in the presence of abnormal CAG repeats. Conclusion: Even though in the last decades many researchers have focused on the development of curative treatment, there is no effective therapeutic target to prevent the clinical onset of the disease or to delay its progression. Instead, current pharmacological management is palliative, the evidence to generalize the surgical approaches is insufficient. Newly, the fetal neural cell transplantation into the striatum is offered as the surgical option that provides hope for the development of a true disease-modifying treatment.

Current Knowledge and Future Directions in Huntington’s Disease

Introduction: Huntington's Disease (HD) is a hereditary, neurodegenerative disorder due to abnormal repeats of the CAG triplet in the IT-15 gene. It is characterized by a triad of progressive motor, psychiatric and cognitive symptoms, resulting from striatal neuronal loss. The impact of HD in Latin America is evidenced by the prevalence rates in Maracaibo (Venezuela) and Juan de Acosta (Colombia), which are the highest recorded in the world. This contrasts with the social abandonment and the scarce intervention of local governments. Aim: Provide an approach to the most relevant aspects of HD from its pathogenesis and associated genetic polymorphisms, to the current treatment options. Methodology: A literature review was performed of the state of the art of prognosis and treatment strategies in HD, including the identification of different polymorphic markers in the genes coding for UCHL1, HIP1, PGC1α, GRIK2, TBP, BDNF, among others, and its associations in the evolution of the disease in the presence of abnormal CAG repeats. Conclusion: Even though in the last decades many researchers have focused on the development of curative treatment, there is no effective therapeutic target to prevent the clinical onset of the disease or to delay its progression. Instead, current pharmacological management is palliative, the evidence to generalize the surgical approaches is insufficient. Newly, the fetal neural cell transplantation into the striatum is offered as the surgical option that provides hope for the development of a true disease-modifying treatment.

Consequences of Viral Infection and Cytokine Production During Pregnancy on Brain Development in Offspring.

Infections during pregnancy can seriously damage fetal neurodevelopment by aberrantly activating the maternal immune system, directly impacting fetal neural cells. Increasing evidence suggests that these adverse impacts involve alterations in neural stem cell biology with long-term consequences for offspring, including neurodevelopmental disorders such as autism spectrum disorder, schizophrenia, and cognitive impairment. Here we review how maternal infection with viruses such as Influenza A, Cytomegalovirus, and Zika during pregnancy can affect the brain development of offspring by promoting the release of maternal pro-inflammatory cytokines, triggering neuroinflammation of the fetal brain, and/or directly infecting fetal neural cells. In addition, we review insights into how these infections impact human brain development from studies with animal models and brain organoids. Finally, we discuss how maternal infection with SARS-CoV-2 may have consequences for neurodevelopment of the offspring.

Effect of various origins conditioned media on the migration of neural cells in vitro

An important direction in the development of the latest technologies for the restoration of damaged central nervous system is the use of stem/progenitor cells (SPCs), mainly neurogenic SPCs (NSPCs) and mesenchymal multipotent stromal cells (MMSCs). One of the main mechanisms of SPCs action is indirect paracrine effects due to the ability to produce a wide range of biologically active signaling molecules (secretome). The study of regenerative effects of conditioned media (CM) of NSPCs and MMSCs as a source of their secretome seems to be actual and potentially beneficial. The aim of the study is to compare the impact of CM from 24-h cultures of fetal neurogenic cells (NCs (E14), as a source of NSPCs) and adiposederived mononuclear cells (AMCs as a source of MMSCs) on migration capacity of rat neural cells in vitro. AMCs-CM were obtained from 24-h cultures with prevalence of CD105+ cells and ability upon further cultivation to form “spheroids” and potency to differentiate into different cell types. NCs-CM were obtained from 24-h cultures with prevalence of Nestin+ cells and ability upon further cultivation to form “neurospheres” and potency to differentiate into astrocytes (GFAP+) and neurons (β-Tubulin III+). Rat fetal neural cells (E14) were cultured to achieve a confluent monolayer with basic cellular elements of nervous tissue (5-7th day), which was dissected with forming a transection site and DMEM with 10% fetal calf serum (control) or 0.1-0.3 mg/ml (by total protein amount) of NCs-CM or AMCs-CM were added. In control cultures of rat neural cells partial overgrowth of the dissected area of the monolayer was observed due to the migration of cells, formation of a network of processes and intercellular contacts; reaching 13.2% (4th day) – 23.2% (8th day) of its full length. The overgrown area increased after addition of CM: NCsCM – 3 times (0.1-0.2 mg/ml) and 3-4 times (0.3 mg/ml, 4th-8th day), reaching 70.5% of full length of the transection site; AMCs-CM – 1.5 times (0.1-0.2 mg/ml) and 4-7 times (0.3 mg/ml, 4th-8th day), reaching 97.4-100% of full length of the transection site. The addition of NCs CM and AMCs CM resulted in β-catenin translocation into nucleus of cells in rat neural cell cultures, which correlated with the overgrowth of the transection zone. NCs-CM as well as AMCs-CM in dose-dependent manner stimulate migration processes in culture of rat neural cells, obviously, involving β-catenin signaling pathway, contributing to overgrowing of the dissected area (reparation of a mechanical defect). NCs-CM and AMCs-CM are a source of signaling molecules that modulate the microenvironment and activate endogenous repair mechanisms in culture (in vitro model of nerve tissue regeneration).

The effects of fetal neural cell conditioned medium on cell proliferation in the rat brain after traumatic brain injury

Traumatic brain injury (TBI) is accompanied by an increase in the number of proliferating cells. However, the question of the nature, conditions of production and mechanisms of action of humoral factors secreted by fetal neural cells (FNCs) on reparative processes and neurogenesis in the brain after trauma and FNCs transplantation remains open. The purpose of the study was to establish the possibility of the influence of the conditioned medium of fetal neural cell cultures on the proliferative activity of Ki-67-positive cells in the cortex and subcortical structures of the rat brain after TBI. Materials and methods. TBI was simulated by dropping a metal cylinder on the rat’s head. Rats (E17-18) were used to obtain cultures of neural stem/progenitor cells. Conditioned media from cell cultures with high adhesive properties (HA-CM) and low adhesive properties (LA-CM) were used to treat the effects of experimental TBI in rats by intramuscular injection. The effect of conditioned media on the proliferative activity of Ki-67-positive cells in the cortex and subcortical structures of the brain after TBI was determined by immunohistochemical analysis using antibodies against Ki-67 protein. Results. Immunohistochemical analysis of the brain sections showed that on the 5th day after traumatic brain injury in rats there was a probable increase in the number of Ki-67-positive cells in the cortex, hippocampus and thalamus. It was found that the injection of HA-CM or LA-CM in animals with TBI increased the number of Ki-67-positive cells in the hippocampus compared with the TBI group and their value for the TBI+LA-CM group reached 59.6 ± 6.1, and for the TBI+HA-CM group – 47.2 ± 3.1 cells (p &lt;0.05 compared with the TBI group). In the cortex and thalamus, the number of Ki-67-positive cells in contrast decreased compared with the group of animals with TBI and for the group TBI+LA-CM was 20.2 ± 1.6 and 12.0 ± 1.7, respectively, and for the group TBI+HA-CM – 25.3 ± 2.1 and 13.3 ± 1.3, respectively. Conclusions. The administration of LA-CM or HA-CM to animals with traumatic brain injury increases the number of Ki-67-positive cells in the hippocampus, possibly associated with increased neurogenesis, and decreases in the cortex and thalamus, which may be due to a weakening of reactive gliosis.

Culturing Mouse Fetal Neural Precursor Cells in a Free-Floating Serum-Free Condition.

Neural precursor cells (NPCs) are a renewable cell source that can proliferate and expand for long periods of time and give rise to the main neural cell types of the central nervous system (CNS). Establishing simple and reproducible growth culture conditions is of great importance to study the biology of NPCs and to understand the molecular basis of their behavior in healthy and diseased conditions.Here, we describe a simple free-floating , serum-free culture condition, known as the neurosphere assay, which is the most commonly used method for the isolation and expansion of NPCs harvested from the adult and fetal CNS. This culture system will result in large numbers of undifferentiated NPC progenies that represents a useful cell source for many in vitro and in vivo applications.

The effects of the conditioned medium of fetal neural cell culture on the apoptosis in the rat brain after traumatic brain injury

A promising treatment method of the traumatic brain injury (TBI) may be stem cell therapy. However, the question of the nature and mechanisms of action of humoral factors produced by stem cells on apoptotic and reparative processes in the brain after trauma remains open. The purpose of the study was to research the effect of conditioned media of fetal neural cell cultures on the number of apoptotic cells in the cortex and subcortical structures of the rat brain after TBI. Materials and methods. TBI was modelled by dropping a metal cylinder on rat's head. Rat fetuses (E17-18) brain was used to obtain cultures of neural stem/progenitor cells. Conditioned media from cell cultures with high adhesive properties (HA-CM) and low adhesive properties (LA-CM) were used to treat the experimental TBI in rats. The presence of p53-positive cells in the cortex and subcortical structures was investigated by immunohistochemistry. Results. Immunohistochemical analysis of brain sections showed that on the 5th day after TBI in rats there was an increase in the number of p53-positive cells in both the cortex and subcortical structures of the brain. Injection of HA-CM and LA-CM to animals on the 2nd, 3rd, 4th days after TBI was found to reduce the number of p53-positive cells in the cortex, hippocampus and thalamus by approximately half compared to the TBI group. A significant difference in the inhibitory effect of two different conditioned media (HA-CM and LA-CM) on apoptosis in the brain of rats after TBI was not detected. Conclusions. The administration of conditioned media of rat fetal neural cell cultures caused a significant decrease in the number of p53-positive cells in both the cortex and subcortical structures on the 5th day after the brain injury.

The efficacy of fetal neural cell aggregates and their combination with fetal liver stromal cells to reduce brain damage after intracerebral hemorrhage in rats

Patients with intracerebral hemorrhage have frequent complications and high mortality. There are currently no effective treatments for this disease. We investigated the effect of the use of cryopreserved aggregates of neural cells in combination with fetal liver stromal cells on the reduction of rat brain injury after intracerebral hemorrhage. Methods. Intracerebral hemorrhage (ICH) was modeled in rats by stereotactic administration of 0.2 U of collagenase type IV into the striatum. Neural cell obtained from brain and stromal cells (SCs) – from liver of rat fetuses of 15 dpc. The suspension of neural cell aggregates (NCAs) alone or in combination with fetal liver stromal cells was injected into the lateral ventricle. The level of lipid peroxidation was determined by the thiobarbituric acid test. The degree of brain cells injury after ICH was determined by the activity of lactate dehydrogenase in blood serum. To assess the intensity of adverse factors and the regenerative potential of different variants of cell therapy, the area of the lost striatum in the rat brain and the average distance from the border of the lesion to the nearest neurons were determined. Results. Combined transplantation of NCAs with fetal liver SCs in rats with ICH was found to reduce malonic dialdehyde concentration and lactate dehydrogenase activity more effectively than NCAs alone, indicating inhibition of lipid peroxidation and reduction of cell injury after intracerebral hemorrhage as a result of the addition of SCs. It was shown a significant decrease in the area of lost striatum in both experimental groups. The single administration of NCAs reduced the distance from the lesion border to the nearest neurons the most, indicating the best conditions for survival and/or regeneration of neurons close to the lesion compared to controls. Conclusions. Administration of NCAs, both alone and in combination with fetal liver SCs, reduces the intensity of oxidative stress, preserves the intact striatum tissue, and increases the number of neurons near the brain lesion in intracerebral hemorrhage in rats. The co-transplantation of fetal liver SCs helps to inhibit lipid peroxidation more effectively.

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures

High-Content Screening Differentiation and Maturation Analysis of Fetal and Adult Neural Stem Cell-Derived Oligodendrocyte Precursor Cell Cultures.

The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation. In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity. Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs. This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.

Immunoregulatory effect of mouse fetal neural cells on the graft-versus-host disease

The problem of the treatment of acute and chronic graft-versus-host disease (GVHD), when histoincompatible bone marrow (BM) is used, remains unsolved. An important role in controlling the development of GVHD is played by Treg immunity.The purpose of the study is to evaluate the immunoregulatory effect of native and cryopreserved murine fetal neural cells (FNCs) relative to Treg immunity of mice with GVHD.Materials and methods. Acute GVHD was induced by the injection of histoincompatible BM to lethally irradiated mice. On the 14th day after GVHD induction and transplantation of native or cryopreserved FNCs in animals of all experimental groups, the spleen index, the content of T-regulatory (FOXP3+) cells and the number of foxp3 gene transcripts in the СD4+splenocytes were determined.Results. The recipients of the histoincompatible BM had a decrease in the content of T-reg cells and the level of foxp3 gene expression in the splenocyte population relative to the syngeneic control. Injection of native or cryopreserved FNCs to animals with GVHD caused an increase in the number of T-reg cells. Cryopreserved FNCs are more than native ones enhancing both the relative number of T-reg cells and the level of foxp3 gene expression in the splenocytes, which was characterized by a higher recipients’ survival up to the 16th day of observation.Conclusion. The transplantation of fetal neural cells to recipients with GVHD stimulates the Treg immunity, which is a key to the development of immune conflict. This confirms the possibility of using fetal neural cells as a therapeutic immuno-regulatory agent.

Isolation and culture of porcine primary fetal progenitors and neurons from the developing dorsal telencephalon.

The development of long-term surviving fetal cell cultures from primary cell tissue from the developing brain is important for facilitating studies investigating neural development and for modelling neural disorders and brain congenital defects. The field faces current challenges in co-culturing both progenitors and neurons long-term. Here, we culture for the first time, porcine fetal cells from the dorsal telencephalon at embryonic day (E) 50 and E60 in conditions that promoted both the survival of progenitor cells and young neurons. We applied a novel protocol designed to collect, isolate and promote survival of both progenitors and young neurons. Herein, we used a combination of low amount of fetal bovine serum, together with pro-survival factors, including basic fibroblast growth factor and retinoic acid, together with arabinofuranosylcytosine and could maintain progenitors and facilitate in vitro differentiation into calbindin 1+ neurons and reelin+ interneurons for a period of 7 days. Further improvements to the protocol that might extend the survival of the fetal primary neural cells would be beneficial. The development of new porcine fetal culture methods is of value for the field, given the pig's neuroanatomical and developmental similarities to the human brain.

Neurorestorative effect of olfactory ensheathing cells and Schwann cells by intranasal delivery for patients with ischemic stroke: design of a multicenter randomized double-blinded placebo-controlled clinical study

Introduction:There have been many clinical studies or trials for patients with ischemic stroke by cell therapy, which includes olfactory ensheathing cell (OEC), mononuclear cell, mesenchymal stromal cell, fetal neural cell or products of varying stem cells, etc. Those cells through different transplanting ways have showed moderate neurorestorative effect in patients with ischemic stroke, but majority were not multicenter randomized, double-blinded, placebo-controlled studies or trials. OEC transplantation has shown a more effective to restore neurological damage in central nervous system (CNS). We hypothesize that OEC through intra-olfactory mucosa transplantation can migrate into the ischemic stroke area around and restore neurological deficit caused from this disaster.Objective of the study:This is a multicenter, randomized, double-blinded, placebo- controlled 12 month clinical study of OECs and Schwann cells (SCs) for patients with sub-acute ischemic stroke and chronic ischemic stroke, to test which kind of cell has more neurorestorative effect for patients with ischemic stroke relative to placebo.Design of the study:This study is involved two groups of patients with sub-acute ischemic stroke and chronic ischemic stroke. Each group enrolls 30 patients. The experimental intervention consists in using OECs and SCs through intra-olfactory mucosa transplantation in participating patients. This will be compared with using placebo (injecting cell culture medium). Participating patients in groups of sub-acute ischemic stroke and chronic ischemic stroke are randomized in natural order to divide into A, B, or C groups and get one of experimental treatment procedures. Patients, operating physicians, and assessing physicians are left unaware of what cells or medium will be injected to participating patients. All patients will be assessed before treatment and after one month, three months, six months, and one year.Ethics and dissemination:The clinical study protocol and consent form were approved by Chinese Association of Neurorestoratology and the ethics committees of the hospitals which joined this clinical study. Findings will be published in peer-reviewed journals.

Dual Effects of Human Placenta-Derived Neural Cells on Neuroprotection and the Inhibition of Neuroinflammation in a Rodent Model of Parkinson’s Disease

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disease in the elderly and the patients suffer from uncontrolled movement disorders due to loss of dopaminergic (DA) neurons on substantia nigra pars compacta (SNpc). We previously reported that transplantation of human fetal midbrain-derived neural precursor cells restored the functional deficits of a 6-hydroxy dopamine (6-OHDA)-treated rodent model of PD but its low viability and ethical issues still remain to be solved. Albeit immune privilege and neural differentiation potentials suggest mesenchymal stem cells (MSCs) from various tissues including human placenta MSCs (hpMSCs) for an alternative source, our understanding of their therapeutic mechanisms is still limited. To expand our knowledge on the MSC-mediated PD treatment, we here investigated the therapeutic mechanism of hpMSCs and hpMSC-derived neural phenotype cells (hpNPCs) using a PD rat model. Whereas both hpMSCs and hpNPCs protected DA neurons in the SNpc at comparable levels, the hpNPC transplantation into 6-OHDA treated rats exhibited longer lasting recovery in motor deficits than either the saline or the hpMSC treated rats. The injected hpNPCs induced delta-like ligand (DLL)1 and neurotrophic factors, and influenced environments prone to neuroprotection. Compared with hpMSCs, co-cultured hpNPCs more efficiently protected primary neural precursor cells from midbrain against 6-OHDA as well as induced their differentiation into DA neurons. Further experiments with conditioned media from hpNPCs revealed that the secreted factors from hpNPCs modulated immune responses and neural protection. Taken together, both DLL1-mediated contact signals and paracrine factors play critical roles in hpNPC-mediated improvement. First showing here that hpMSCs and their neural derivative hpNPCs were able to restore the PD-associated deficits via dual mechanisms, neuroprotection and immunosuppression, this study expanded our knowledge of therapeutic mechanisms in PD and other age-related diseases.

In vivo transplantation of fetal human gut-derived enteric neural crest cells.

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75NTR were lentivirally labeled with either GFP or calcium‐sensitive GCaMP and transplanted into the hindgut of Rag2 − /γc − /C5 −‐immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut‐derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.