Immunocytochemical Localization Research Articles

Exploration of immunomodulatory mechanism of caprine Wharton’s jelly derived mesenchymal stem cells

The present study was aimed to explore the possible mechanisms by which caprine Wharton’s jelly-derived MSCs (WJ-MSCs) perform their immunomodulatory function. WJ-MSCs were isolated through explants culture and characterized as per ISCT criteria using culture behavior, expression of surface markers by PCR, FACS and immunocytochemical localization (ICC), trilineage differentiation potential etc. Secretory behavior for important biomolecules (IDO, TGFβ1, VEGF, IL6) was evaluated by ICC and western blot assay. Cell-to-cell communication was studied by culturing cells in cell–cell contact and trans-well system. The MSCs when co-cultured with activated Tc and Th cells, down-regulation of T cell cytokine as well as upregulation of immunomodulatory factors (VEGF A, IL10, IL6, IDO, iNOS, PTGS2, HGF, TGFβ, CXCL10, CXCL11) was noticed in both cell–cell contact and trans-well culture system which was significantly higher in cell–cell contact system. Trilineage differentiation of MSCs showed significant upregulation of MHC I (CAHI) and MHC II (CLA DRB3) molecules suggesting better clinical applications of MSCs without differentiation to avoid immune rejection. It can be concluded that WJ-MSCs perform their immunomodulation through the secretion of a battery of biomolecules and work in both cell–cell contact manner and through their secretome.

CgLS mediates limonene synthesis of main essential oil component in secretory cavity cells of Citrus grandis ‘Tomentosa’ fruits

d-Limonene is the predominant component of essential oil from Exocarpium Citri Grandis, known for its antibacterial, antioxidant, insecticidal, and anti-inflammatory properties. The synthesis, transport, and accumulation of d-limonene in Citrus grandis ‘Tomentosa’ fruits are regulated by limonene synthase (LS) and its associated regulatory genes. This study addresses the gap in understanding the spatiotemporal cytological changes of LS and its regulatory genes. Through cytochemical techniques, we investigated the distribution of essential oil in the plastids, endoplasmic reticulum, and vacuoles of secretory cavity cells. We identified the LS-encoding gene CgLS via transcriptomics and demonstrated in vitro that CgLS catalyzes the formation of d-limonene from geranyl diphosphate (GPP). Transient overexpression of CgLS increased monoterpene limonene accumulation, while TRV virus-induced gene silencing reduced it. CgLS expression levels and d-limonene content showed spatiotemporal consistency with fruit development, with in situ hybridization revealing predominant expression in secretory cavity cells. Immunocytochemical localization indicated that CgLS is primarily located in the endoplasmic reticulum, plastids, and vacuoles. Our findings suggest that CgLS is translated in the endoplasmic reticulum and transported to plastids and vacuoles where d-limonene synthesis occurs. This study provides comprehensive insights into the characteristics of CgLS and its role in d-limonene synthesis at the tissue, cellular, and molecular levels in C. grandis ‘Tomentosa’.

Plant-specific calreticulin is localized in the nuclei of highly specialized cells in the pistil-new observations for an old hypothesis.

One of the first cellular locations of the calreticulin (CRT) chaperone in eukaryotic cells, apart from its obvious localization in the endoplasmic reticulum (ER), was the cell nucleus (Opas et al. 1991). The presence of CRT has been detected inside the nucleus and in the nuclear envelope of animal and plant cells, and a putative nuclear localization signal (NLS) in the CRT amino acid sequence has been mapped in several animal and plant species. Over the last 30years, other localization sites of this protein outside the ER and cell nucleus have also been discovered, suggesting that CRT is a multifunctional Ca2+-binding protein widely found in various cell types. In our previous studies focusing on plant developmental biology, we have demonstrated the presence of CRT inside and outside the ER in highly specialized plant cells, as well as the possibility of CRT localization in the cell nucleus. In this paper, we present a detailed analysis of immunocytochemical localization of CRT inside nuclei of the pistil transmission tract somatic cells before and after pollination. We show a similar pattern of the nuclear CRT localization in relation to exchangeable Ca2+ for two selected species of angiosperms, dicotyledonous Petunia and monocot Haemanthus, that differ in anatomical structure of the pistil and discuss the potential role of CRT in the cell nucleus.

Three-dimensional analysis ofthe intracellular architecture by scanning electron microscopy.

The two-dimensional observation of ultrathin sections from resin-embedded specimens provides an insufficient understanding of the three-dimensional (3D) morphological information of membranous organelles. The osmium maceration method, developed by Professor Tanaka's group >40 years ago, is the only technique that allows direct observation of the 3D ultrastructure of membrane systems using scanning electron microscopy (SEM), without the need for any reconstruction process. With this method, the soluble cytoplasmic proteins are removed from the freeze-cracked surface of cells while preserving the integrity of membranous organelles, achieved by immersing tissues in a diluted osmium solution for several days. By employing the maceration method, researchers using SEM have revealed the 3D ultrastructure of organelles such as the Golgi apparatus, mitochondria and endoplasmic reticulum in various cell types. Recently, we have developed new SEM techniques based on the maceration method to explore further possibilities of this method. These include: (i) a rapid osmium maceration method that reduces the reaction duration of the procedure, (ii) a combination method that combines agarose embedding with osmium maceration to elucidate the 3D ultrastructure of organelles in free and cultured cells and (iii) a correlative immunofluorescence and SEM technique that combines cryosectioning with the osmium maceration method, enabling the correlation of the immunocytochemical localization of molecules with the 3D ultrastructure of organelles. In this paper, we review the novel osmium maceration methods described earlier and discuss their potential and future directions in the field of biology and biomedical research.

Ca2+- and Zn2+-dependent nucleases co-participate in nuclear DNA degradation during programmed cell death in secretory cavity development in Citrus fruits.

Calcium (Ca2+)- and zinc Zn2+-dependent nucleases play pivotal roles in plant nuclear DNA degradation in programmed cell death (PCD). However, the mechanisms by which these two nucleases co-participate in PCD-associated nuclear DNA degradation remain unclear. Here, the spatiotemporal expression patterns of two nucleases (CrCAN and CrENDO1) were analyzed qualitatively and quantitatively during PCD in secretory cavity formation in Citrus reticulata 'Chachi' fruits. Results show that the middle and late initial cell stages and lumen-forming stages are key stages for nuclear degradation during the secretory cavity development. CAN and ENDO1 exhibited potent in vitro DNA degradation activity at pH8.0 and pH5.5, respectively. Quantitative real-time reverse-transcription polymerase chain reaction, in situ hybridization assays, the subcellular localization of Ca2+ and Zn2+, and immunocytochemical localization showed that CrCAN was activated at the middle and late initial cell stages, while CrENDO1 was activated at the late initial cell and lumen-forming stages. Furthermore, we used immunocytochemical double-labelling to simultaneously locate CrCAN and CrENDO1. The DNA degradation activity of the two nucleases was verified by simulating the change of intracellular pH in vitro. Our results also showed that CrCAN and CrENDO1 worked respectively and co-participated in nuclear DNA degradation during PCD of secretory cavity cells. In conclusion, we propose the model for the synergistic effect of Ca2+- and Zn2+-dependent nucleases (CrCAN and CrENDO1) in co-participating in nuclear DNA degradation during secretory cavity cell PCD in Citrus fruits. Our findings provide direct experimental evidence for exploring different ion-dependent nucleases involved in nuclear degradation during plant PCD.

Decidual lymphatic endothelial cell-derived granulocyte-macrophage colony-stimulating factor induces M1 macrophage polarization via the NF-κB pathway in severe pre-eclampsia.

Direct interactions between macrophages and lymphatic vessels have been shown previously.In pre-eclampsia (PE), macrophages are dominantly polarized into a proinflammatory M1 phenotype and lymphangiogenesis is defective in the decidua.Here, we investigated whether decidual lymphatic endothelial cells (dLECs) affect macrophage polarization in PE. THP-1 macrophages were cocultured with dLECs or cultured in the conditioned medium (CM) of dLECs. Macrophage polarization was measured using flow cytometry. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in dLECs was measured using qRT-PCR and ELISA. The activation of nuclear translocation of nuclear factor-κ (NF-κB), an upstream signaling molecule of GM-CSF, was assessed by immunocytochemical localization of p65. Through GM-CSF knockdown and NF-κB inhibition in dLEC, we evaluated whether the GM-CSF/NF-κB pathway of PE dLEC affects decidual macrophage polarization. The ratio of inflammatory M1 macrophages with HLA-DR+ /CD80+ markers significantly increased following coculturing with PE dLECs or culturing in PE dLEC CM, indicating that the PE dLEC-derived soluble factor acts in a paracrine manner. GM-CSF expression was significantly upregulated in PE dLECs. Recombinant human GM-CSF induced macrophage polarization toward an M1-like phenotype, whereas its knockdown in PE dLECs suppressed it, suggesting PE dLECs induce M1 macrophage polarization by secreting GM-CSF. The NF-κB p65 significantly increased in PE dLECs compared to the control, and pretreatment with an NF-κB inhibitor significantly suppressed GM-CSF production from PE dLECs. In PE, dLECs expressing high levels of GM-CSF via the NF-κB-dependent pathway play a role in inducing decidual M1 macrophage polarization.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Evidence that Nectin-3 is the soybean agglutinin binding protein on rat corneal endothelium cell surfaces

The means by which the lectin soybean agglutinin (SBA) binds to the corneal endothelium cell surface following explantation into organ culture was investigated using Sprague-Dawley rats. SBA binding does not occur in freshly isolated and fixed rat corneal endothelium. However, after 48 h in organ culture, SBA binding occurs in a punctate pattern that clearly outlines all endothelial cells of the tissue monolayer. To determine what cell surface component was responsible for this binding, a series of experiments were employed that focused on the possibility that SBA bound to a nectin molecule(s). To this extent we performed a series of immunocytochemical localizations using antibodies against either nectin-2, nectin-3 or nectin-4. Of these, only nectin-3 bound to the endothelium in a manner that mimicked SBA binding. To further verify that nectin-3 bound SBA, displacement experiments employing non-labeled SBA were undertaken. Following a 48 h organ culture, tissues were fixed and incubated with SBA followed by exposure to nectin-3 antibody. No subsequent immunofluorescence could be detected, indicating that anti-nectin-3 binding was prevented. Likewise, when organ-cultured tissues were fixed and incubated in anti-nectin-3 antibody, followed by SBA exposure, no SBA binding could be detected. These results suggest that stresses accompanying explantation of the tissue into organ culture promote the appearance of nectin-3 around the cell periphery. The emergence of nectin-3 along the peripheral endothelial cell membrane in organ culture may imply a necessary role for this molecule in maintaining monolayer integrity and barrier function during either a pathologic condition, wound repair, or in organ storage.

Immunocytochemical localization of the AMPA glutamate receptor subtype GluR2/3 in the squid optic lobe

As a major excitatory neurotransmitter in the cephalopod visual system, glutamate signaling is facilitated by ionotropic receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR). In cephalopods with large and well-developed brains, the optic lobes (OL) mainly process visual inputs and are involved in learning and memory. Although the presence of AMPAR in squid OL has been reported, the organization of specific AMPAR-containing neurons remains unknown. This study aimed to investigate the immunocytochemical localization of the AMPA glutamate receptor subtype 2/3-immunoreactive (GluR2/3-IR) neurons in the OL of Pacific flying squid (Tordarodes pacificus). Morphologically diverse GluR2/3-IR neurons were predominantly located in the tangential zone of the medulla. Medium-to-large GluR2/3-IR neurons were also detected. The distribution patterns and cell morphologies of calcium-binding protein (CBP)-IR neurons, specifically calbindin-D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-IR neurons, were similar to those of GluR2/3-IR neurons. However, two-color immunofluorescence revealed that GluR2/3-IR neurons did not colocalize with the CBP-IR neurons. Furthermore, the specific localizations and diverse types of GluR2/3-IR neurons that do not express CB, CR, or PV in squid OL were determined. These findings further contribute to the existing data on glutamatergic visual systems and provide new insights for understanding the visual processing mechanisms in cephalopods.

Immunocytochemical Localization of Enzymes Involved in Dopamine, Serotonin, and Acetylcholine Synthesis in the Optic Neuropils and Neuroendocrine System of Eyestalks of Paralithodes camtschaticus.

Identifying the neurotransmitters secreted by specific neurons in crustacean eyestalks is crucial to understanding their physiological roles. Here, we combined immunocytochemistry with confocal microscopy and identified the neurotransmitters dopamine (DA), serotonin (5-HT), and acetylcholine (ACh) in the optic neuropils and X-organ sinus gland (XO-SG) complex of the eyestalks of Paralithodes camtschaticus (red king crab). The distribution of Ach neurons was studied by choline acetyltransferase (ChAT) immunohistochemistry and compared with that of DA neurons examined in the same or adjacent sections by tyrosine hydroxylase (TH) immunohistochemistry. We detected 5-HT, TH, and ChAT in columnar, amacrine, and tangential neurons in the optic neuropils and established the presence of immunoreactive fibers and neurons in the terminal medulla in the XO region of the lateral protocerebrum. Additionally, we detected ChAT and 5-HT in the endogenous cells of the SG of P. camtschaticus for the first time. Furthermore, localization of 5-HT- and ChAT-positive cells in the SG indicated that these neurotransmitters locally modulate the secretion of neurohormones that are synthesized in the XO. These findings establish the presence of several neurotransmitters in the XO-SG complex of P. camtschaticus.

PFTAIRE Kinase L63 Interactor 1A (Pif1A Protein) Is Required for Actin Cone Movement during Spermatid Individualization in Drosophila melanogaster.

A useful model for determining the mechanisms by which actin and actin binding proteins control cellular architecture is the Drosophila melanogaster process of spermatogenesis. During the final step of spermatogenesis, 64 syncytial spermatids individualized as stable actin cones move synchronously down the axonemes and remodel the membranes. To identify new genes involved in spermatid individualization, we screened a collection of Drosophila male-sterile mutants and found that, in the line Z3-5009, actin cones formed near to the spermatid nuclei but failed to move, resulting in failed spermatid individualization. However, we show by phalloidin actin staining, electron microscopy and immunocytochemical localization of several actin binding proteins that the early cones had normal structure. We sequenced the genome of the Z3-5009 line and identified mutations in the PFTAIRE kinase L63 interactor 1A (Pif1A) gene. Quantitative real-time PCR showed that Pif1A transcript abundance was decreased in the mutant, and a transgene expressing Pif1A fused to green fluorescent protein (GFP) was able to fully rescue spermatid individualization and male fertility. Pif1A-GFP localized to the front of actin cones before initiation of movement. We propose that Pif1A plays a pivotal role in directing actin cone movement.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Immunocytochemical Localization of XRCC1 and γH2AX Foci Induced by Tightly Focused Femtosecond Laser Radiation in Cultured Human Cells.

To assess the prospects for using intense femtosecond laser radiation in biomedicine, it is necessary to understand the mechanisms of its action on biological macromolecules, especially on the informational macromolecule—DNA. The aim of this work was to study the immunocytochemical localization of DNA repair protein foci (XRCC1 and γH2AX) induced by tightly focused femtosecond laser radiation in human cancer A549 cells. The results showed that no XRCC1 or γH2AX foci tracks were observed 30 min after cell irradiation with femtosecond pulses of 1011 W∙cm−2 peak power density. An increase in the pulse power density to 2 × 1011 W∙cm−2 led to the formation of linear tracks consisting both of XRCC1 and γH2AX protein foci localized in the places where the laser beam passed through the cell nuclei. A further increase in the pulse power density to 4 × 1011 W∙cm−2 led to the appearance of nuclei with total immunocytochemical staining for XRCC1 and γH2AX on the path of the laser beam. Thus, femtosecond laser radiation can be considered as a tool for local ionization of biological material, and this ionization will lead to similar effects obtained using ionizing radiation.

Immunocytochemical localization of leptin hormone in the neurosecretory cells of brain–suboesophageal ganglion complex of tropical tasar silkworm, Antheraea mylitta (D.) eco-race Bhandara

Immunocytochemical localization of leptin hormone in the neurosecretory cells of brain–suboesophageal ganglion complex of tropical tasar silkworm, Antheraea mylitta (D.) eco-race Bhandara

Histology and Immunocytochemical Localization of Glial Fibrillary Acidic Protein in the Inner Ear of Scincella tsinlingensis

Histology and Immunocytochemical Localization of Glial Fibrillary Acidic Protein in the Inner Ear of Scincella tsinlingensis

Characterization of Antennal Sensilla and Immunolocalization of Odorant-Binding Proteins on Spotted Alfalfa Aphid, Therioaphis trifolii (Monell).

The spotted alfalfa aphid [Therioaphis trifolii (Monell), Homoptera, Drepanosiphidae] is a well-known destructive pest that can significantly reduce alfalfa yields. Herein, the morphology of antennal sensilla of T. trifolii has been examined by using scanning electron microscopy and the ultrastructure of sensilla stellate and placoidea was described by transmission electron microscopy. Stellate sensilla, placoid sensilla, and coeloconic sensilla were found on the 6th segment, and a single sensillum placoidea was located on the 5th segment. Placoid sensilla were also present on the 3rd antennal segment of alate and apterous aphids, and the number was similar between two morphs. Two types of trichoid sensilla and coeloconic sensilla were found on the antennae, respectively. The results of ultrastructure showed that stellate sensilla are innervated by three neurons, while placoid sensilla present three groups of neurons, equipped with 2–3 dendrites in each neuron group. Immunocytochemical localization of odorant-binding proteins (OBPs) was performed on ultrathin sections of sensilla stellate and placoidea, and we observed that the antiserum against OBP6 intensively labeled all placoid sensilla from both primary and secondary rhinaria. OBP7 and OBP8 could also be detected in placoid sensilla, but less strongly than OBP6. In addition, OBP6, OBP7, and OBP8 were densely labeled in stellate sensilla, suggesting OBP6, OBP7, and OBP8 may sense alarm pheromone germacrene A in T. trifolii.

Immunocytochemical localization of tyrosine hydroxylase in the visual cortex of the microbat, Rhinolophus ferrumequinum.

In order to enhance our understanding of bat vision, we investigated tyrosine hydroxylase (TH)-immunoreactive (IR) fibers in the visual cortex of the microbat. The study was conducted on 12 freshly-caught adult bats (Rhinolophus ferrumequinum, both sexes, weighing 15-20 g). We used standard immunocytochemistry and confocal microscopy. TH-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer I. Two types of TH-IR fibers were observed: small and large varicose fibers. TH-IR cells were not found in the microbat visual cortex. The microbat substantia nigra and ventral tegmental areas, previously identified sources of TH-IR fibers in the mammalian visual cortex, all contained strongly labeled TH-IR cells. The average diameters of TH-IR cells in the substantia nigra and the ventral tegmental areas were 14.39 ± 0.13 μm (mean ± SEM) and 11.85 ± 0.13 μm, respectively. Our results suggest that the microbat has a well-constructed neurochemical organization of THIR fibers. This observation should provide fundamental insights into a better understanding of the nocturnal, echolocating bat visual system.

Immunocytochemical Localization of Olfactory-signaling Molecules in Human and Rat Spermatozoa.

Expression of olfactory receptors (ORs) in non-olfactory tissues has been widely reported over the last 20 years. Olfactory marker protein (OMP) is highly expressed in mature olfactory sensory neurons (mOSNs) of the olfactory epithelium. It is involved in the olfactory signal transduction pathway, which is mediated by well-conserved components, including ORs, olfactory G protein (Golf), and adenylyl cyclase 3 (AC3). OMP is widely expressed in non-olfactory tissues with an apparent preference for motile cells. We hypothesized that OMP is expressed in compartment-specific locations and co-localize with an OR, Golf, and AC3 in rat epididymal and human-ejaculated spermatozoa. We used immunocytochemistry to examine the expression patterns of OMP and OR6B2 (human OR, served as positive olfactory control) in experimentally induced modes of activation and determine whether there are any observable differences in proteins expression during the post-ejaculatory stages of spermatozoal functional maturation. We found that OMP was expressed in compartment-specific locations in human and rat spermatozoa. OMP was co-expressed with Golf and AC3 in rat spermatozoa and with OR6B2 in all three modes of activation (control, activated, and hyperactivated), and the mode of activation changed the co-expression pattern in acrosomal-reacted human spermatozoa. These observations suggest that OMP expression is a reliable indicator of OR-mediated chemoreception, may be used to identify ectopically expressed ORs, and could participate in second messenger signaling cascades that mediate fertility.

Tyrosine Kinase Inhibitors Reduce NMDA NR1 Subunit Expression, Nuclear Translocation, and Behavioral Pain Measures in Experimental Arthritis.

In the lumbar spinal cord dorsal horn, release of afferent nerve glutamate activates the neurons that relay information about injury pain. Here, we examined the effects of protein tyrosine kinase (PTK) inhibition on NMDA receptor NR1 subunit protein expression and subcellular localization in an acute experimental arthritis model. PTK inhibitors genistein and lavendustin A reduced cellular histological translocation of NMDA NR1 in the spinal cord occurring after the inflammatory insult and the nociceptive behavioral responses to heat. The PTK inhibitors were administered into lumbar spinal cord by microdialysis, and secondary heat hyperalgesia was determined using the Hargreaves test. NMDA NR1 cellular protein expression and nuclear translocation were determined by immunocytochemical localization with light and electron microscopy, as well as with Western blot analysis utilizing both C- and N-terminal antibodies. Genistein and lavendustin A (but not inactive lavendustin B or diadzein) effectively reduced (i) pain related behavior, (ii) NMDA NR1 subunit expression increases in spinal cord, and (iii) the shift of NR1 from a cell membrane to a nuclear localization. Genistein pre-treatment reduced these events that occur in vivo within 4 h after inflammatory insult to the knee joint with kaolin and carrageenan (k/c). Cycloheximide reduced glutamate activated upregulation of NR1 content confirming synthesis of new protein in response to the inflammatory insult. In addition to this in vivo data, genistein or staurosporin inhibited upregulation of NMDA NR1 protein and nuclear translocation in vitro after treatment of human neuroblastoma clonal cell cultures (SH-SY5Y) with glutamate or NMDA (4 h). These studies provide evidence that inflammatory activation of peripheral nerves initiates increase in NMDA NR1 in the spinal cord coincident with development of pain related behaviors through glutamate non-receptor, PTK dependent cascades.

Heterogeneous distribution of ion transporters in enteroids enriched in different intestinal epithelial cell types

The small intestine which is characterized by different cell types lining specific sites along the crypt‐villus axis plays a critical role in water, electrolyte and mineral balance. Normally, net fluid absorption by the small intestine occurs mainly because ion‐dependent fluid absorption in the villus column is greater than ion‐driven fluid secretion in the crypt. To generate these site‐specific ion gradients, appropriate transporters must be strategically located in cells lining the crypt or villus regions. The multivalent ions calcium and iron also function as essential minerals, and deficiency in these minerals is highly prevalent worldwide. The crypt‐villus site of many electrolyte and mineral transporters has been localized by immunocytochemistry, but their expression in rare cell types like stem cells (ISC) is unknown. To test the hypothesis that representative ion and mineral transporters are distributed heterogeneously among the various intestinal cell types, we made enteroids enriched in ISC, enterocytes (ENT), goblet (GOB), and Paneth (PAN) cells. We then confirmed by cell‐type specific biomarkers that ISC, ENT, GOB and PAN enteroids were enriched in stem cells (~90% of all cells per organoid), enterocytes (90%), goblet (80%) and Paneth (65%) cells, respectively. ENT and GOB enteroids had the highest mRNA levels of the chloride‐anion exchanger DRA and sodium‐hydrogen exchanger NHE3, while the sodium‐bicarbonate cotransporter NBCe1 was expressed primarily in ENT enteroids. Conversely, the chloride channel CFTR and sodium‐potassium‐chloride cotransporter NKCC1 mRNA were mainly expressed in ISC and PAN enteroids. Findings confirm previous immunocytochemical localization of NBCe1 and DRA in the villus region lined by enterocytes, with CFTR and NKCC1 mainly in intestinal crypts containing Paneth and stem cells. Basolateral NKCC1 staining was observed in ISC and PAN, but not in ENT and GOB, enteroids. The membrane location and expression of NKCC1 in crypt‐dwelling cells reflect its prominent role in intestinal fluid secretion. Members of the intestinal epithelial transport system for iron (DMT1, FTL, IREG1 and HEPH) showed homogeneous distribution in all cell types. The calcium transporters TRPV6 and PMCA1 were expressed least in ENT and GOB, respectively. Our findings depict a mostly heterogeneous distribution of ion transporters in different cell types, reflecting their likely distribution along the crypt villus axis in vivo, suggesting that appropriate expression in different cell types may be essential for ion and mineral homeostasis.Support or Funding InformationSupported by NSF 1456673, 1754783 (RF) and NIH R01AT010243 (RF, NG)