Development of a High-Yield Isolation Protocol Optimized for the Retrieval of Active Muscle Satellite Cells from Mouse Skeletal Muscle Tissue.

Background and Objectives: Difficulties often encountered in separating and purifying active muscle satellite cells (MSCs) from skeletal muscle tissues have limited the supply of cells for muscle therapy and artificial meat production. Here, we report an effective isolation protocol to economically and conveniently retrieve active MSCs from skeletal muscle tissues in mice. Methods and Results: We optimized an enzyme-based tissue digestion protocol for isolating skeletal muscle-derived primary cell population having a large number of active MSCs and described a method of differential plating (DP) for improving purity of active MSCs from skeletal muscle-derived primary cell population. Then, the age of the mouse appropriate to the isolation of a large number of active MSCs was elucidated. The best isolation yield of active MSCs from mouse skeletal muscle tissues was induced by the application of DP method to the primary cell population harvested from skeletal muscle tissues of 2-week-old mice digested in 0.2% (w/v) collagenase type II for 30 min at 37℃ and then in 0.1% (w/v) pronase for 5 min at 37℃. Conclusions: The protocol we developed not only facilitates the isolation of MSCs but also maximizes the retrieval of active MSCs. Our expectation is that this protocol will contribute to the development of original technologies essential for muscle therapy and artificial meat industrialization in the future.

The native form of follicle-stimulating hormone is essential for the growth of mouse preantral follicles in vitro.

To assess whether the follicle-stimulating hormone (FSH) subunits observed in patients with gonadotroph adenomas (GA) can cause infertility, the effects of subunits and heterodimeric FSH on the in vitro follicle development were evaluated in mice. The partial forms of FSH in follicle culture did not induce development into pseudoantral follicles, whereas follicles cultured with native FSH developed into pseudoantral follicles and produced mature metaphase II oocyte. Therefore, intact FSH is needed for folliculogenesis, implying that production of FSH with a partial structure in GA may result in infertility.

Localization of integrin heterodimer α9ß1 on the surface of uterine endometrial stromal and epithelial cells in mice.

Previously, we reported that endometrial stromal (ES) and endometrial epithelial (EE) cells did not attach to tenascin C, indicating the absence of active integrin α9ß1 on the surface of mouse ES and EE cells. However, that study used recombinant tenascin C without fibronectin (FN) type III repeats interacting with integrin heterodimers. Therefore, we re-evaluated the presence of integrin α9ß1 actively functioning on the surface of mouse ES and EE cells using full-length native tenascin C with FN type III repeats. The functionality of integrin α9ß1 was confirmed using attachment and antibody inhibition assays. Both mouse ES and EE cells showed significantly increased adhesion to native tenascin C, and functional blocking of integrin α9ß1 significantly inhibited adhesion to native tenascin C. These results demonstrate that the integrin α9 and ß1 subunits function as active heterodimers on the plasma membrane of mouse ES and EE cells, respectively.

Investigation of Age-Related Changes in the Skin Microbiota of Korean Women.

The microbiota of human skin is influenced by host and environmental factors. To determine if chronological age influences the composition of the skin microbiota on the forehead and hands, 73 Korean women were sorted into one of three age groups: (1) 10-29 years (n = 24), (2) 30-49 years (n = 21), and (3) 50-79 years (n = 28). From the 73 women, 146 skin samples (two skin sites per person) were collected. 16S rRNA gene amplicon sequencing was then conducted to analyze the skin microbiota. The overall microbial distribution varied on the forehead but was similar on the hands across the three age groups. In addition, the composition of the skin microbiota differed between the forehead and hands. Commensal microbiota, such as Streptococcus, Staphylococcus, Cutibacterium, and Corynebacterium, which contribute to maintaining skin health via dominant occupation, were affected by increasing age on forehead and hand skin. Alpha diversity indices increased significantly with age on forehead skin. This study indicates that older people may be more susceptible to pathogenic invasions due to an imbalanced skin microbiota resulting from age-related changes. The results of our study may help develop new strategies to rebalance skin microbiota shifted during aging.

Screening of Integrin Heterodimers Expressed Functionally on the Undifferentiated Spermatogonial Stem Cells in the Outbred ICR Mice.

BACKGROUND AND OBJECTIVES: Outbred mice are widely used in toxicology, pharmacology, and fundamental biomedical research. However, there have been no reports of in vitro culture systems for spermatogonial stem cells (SSCs) derived from these mice. METHODS: As a step towards constructing a non-cellular niche supporting the in vitro maintenance of outbred mouse SSC self-renewal, we systematically investigated the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in SSCs derived from Imprinting Control Region (ICR) mice. RESULTS: Among the genes encoding 25 integrin subunits, integrin α1, α5, α6, α9, αV, and αE, and integrin ß1 and ß5 had significantly higher transcriptional levels than the other subunits. Furthermore, at the translational level, integrin α5, α6, α9, αV, αE, and ß1 were localized on the surface of SSCs, but integrin α1 and ß5 not. Moreover, significantly stronger translational expression than integrin α9 and αE was observed in integrin α5, α6, αV, and ß1. SSCs showed significantly increased adhesion to fibronectin, laminin, tenascin C and vitronectin, and functional blocking of integrin α5ß1, α6ß1, α9ß1 or αVß1 significantly inhibited adhesion to these molecules. CONCLUSIONS: We confirmed that integrin α5ß1, α6ß1, α9ß1 and αVß1 actively function on the surface of undifferentiated SSCs derived from outbred ICR mice.

Generation of embryonic stem cells derived from the inner cell mass of blastocysts of outbred ICR mice.

Embryonic stem cells (ESCs) derived from outbred mice which share several genetic characteristics similar to humans have been requested for developing stem cell-based bioengineering techniques directly applicable to humans. Here, we report the generation of ESCs derived from the inner cell mass of blastocysts retrieved from 9-week-old female outbred ICR mice mated with 9-week-old male outbred ICR mice (ICRESCs). Similar to those from 129/Ola mouse blastocysts (E14ESCs), the established ICRESCs showed inherent characteristics of ESCs except for partial and weak protein expression and activity of alkaline phosphatase. Moreover, ICRESCs were not originated from embryonic germ cells or pluripotent cells that may co-exist in outbred ICR strain-derived mouse embryonic fibroblasts (ICRMEFs) used for deriving colonies from inner cell mass of outbred ICR mouse blastocysts. Furthermore, instead of outbred ICRMEFs, hybrid B6CBAF1MEFs as feeder cells could sufficiently support in vitro maintenance of ICRESC self-renewal. Additionally, ICRESC-specific characteristics (self-renewal, pluripotency, and chromosomal normality) were observed in ICRESCs cultured for 40th subpassages (164 days) on B6CBAF1MEFs without any alterations. These results confirmed the successful establishment of ESCs derived from outbred ICR mice, and indicated that self-renewal and pluripotency of the established ICRESCs could be maintained on B6CBAF1MEFs in culture.

Screening of integrins localized on the surface of human epidermal melanocytes.

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and ß subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, ß1, ß3, ß5, and ß8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, ß1, ß3, and ß5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2ß1 heterodimer and the inactive integrin α4, α5, αV, ß3, and ß5 subunits are all localized on the surface of HEMs.

Recombinant FNIII9-10-derived extracellular signaling effects on the physiology of dermal fibroblasts during in vitro culture.

Previous reports showed that fibronectin (FN) was effective in stimulating the recovery of damaged dermis. However, native FN has multifunctional domains transmitting beneficial as well as unbeneficial signals to dermal tissue cells through the mediation of integrin heterodimers. The use of a functional domain [FN type III9-10 fragments (FNIII9-10)] providing beneficial effects on the physiology of dermal tissue cells would enhance an in vitro culture system for dermal fibroblasts (DFs). We therefore investigated the FNIII9-10-derived extracellular signaling effect on the physiology of DFs during in vitro culture. Recombinant FNIII9-10 proteins were constructed and their functionality was determined by observing the adhesion of adult human DFs (aHDFs) to recombinant FNIII9-10 and of low adhesion integrin α5ß1- and αvß3-blocked aHDFs to recombinant FNIII9-10. Cellular proliferation, morphology, and senescence were measured and compared in the aHDFs cultured on native FN and recombinant FNIII9-10 for short or long periods. The results show that recombinant FNIII9-10-derived extracellular signaling stimulated increased proliferation of aHDF (both in short- and long-term cultures) and inhibited the generation of morphological abnormalities (in short- and long-term cultures) and cellular senescence (long-term culture) when compared with native FN-derived extracellular signaling. Our results suggest that, instead of native FN, recombinant FNIII9-10 better enhanced the in vitro culture of aHDFs while diminishing the adverse effects associated with the use of human-derived materials.

Comparing greenhouse gas emissions and nutritional values based on Korean suggested meal plans and modified vegan meal plans.

Producing animal products from farm to table emits massive amounts of greenhouse gases (GHGs). Modified meal plans, mainly including vegetables and grains, have been recommended to reduce GHG emissions. However, these meal plans have not been developed with regard to the micronutrient content, but rather with regard to the energy requirements of grains and vegetables, which could result in a nutritional imbalance. For this reason, we investigated a common Korean suggested meal plan (SMP) from the National Institute of Agricultural Sciences, in which nutritional conditions were considered, and evaluated its GHG emissions using the Life Cycle Assessment Inventory Database and nutritional values. The SMP, which included meat, was based on the Korean Nutrition Society for adult men age 19 to 29, and was changed to a vegan meal plan (VMP). Animal-based protein sources were substituted for meat alternatives, such as beans and tofu, for which carbon footprint data was available. To compare the nutritional differences, the 9th Korean Food Composition Tables I and II were consulted. To calculate GHG emissions, the carbon footprint data of the food was converted to a CO2 equivalent (CO2e) using a procedure from the Foundation of Agriculture Technology Commercialization and Transfer. It was found that GHG emissions per calorie were 18% lower for the VMP when compared to the SMP. However, if GHG emissions per total amino acids were evaluated, the VMP GHG emissions per total amino acids were 0.12 g CO2e/mg, while the corresponding value for the SMP was 0.06 g CO2e/mg. The Korean daily meat intake reported by the Korea Agricultural Statistics Service was 37.1% lower than in the SMP, but when converted to a protein intake the figure was 17.0% lower. It was found that each SMP resulted in more GHG emissions than the VMP, but when considered as GHG emissions per total amino acids, the opposite pattern was apparent. There is a need to conduct more detailed studies of the variation in GHG emissions with different meal plans, using the daily meat intake per person.

Integrins expressed on the surface of human endometrial stromal cells derived from a female patient experiencing spontaneous abortion.

Here, as a basic study in revealing the correlation between extracellular matrix components and spontaneous abortion, we defined the types of integrins expressed on the surface of endometrial stromal (ES) cells retrieved from the uterus of a patient experiencing spontaneous abortion. For these, the types of integrin subunits in the ES cells retrieved from a woman with spontaneous abortion were identified at the transcriptional and translational levels, and functional assay was conducted for confirming the combinations of integrin α and ß subunits. Among the genes encoding 25 integrin subunits, significantly high transcription was seen in integrins α1, α2, α3, α4, α5, αV, ß1, ß3, and ß5. Translation of integrins α1, α3, α5, αV, and ß1 on the cell surface was detected in almost all ES cells, whereas integrins α2, α4, ß3, and ß4 were expressed translationally only in some ES cells. Subsequently, ES cells showed significantly increased adhesion to collagen I, laminin, fibronectin, and vitronectin, and functional blocking of integrin α1, α3, α5, and αV significantly inhibited adhesion to these molecules. These results demonstrated that active heterodimers composed of integrins α1ß1, α3ß1, α5ß1, and αVß1 were co-localized on the surface of ES cells derived from a patient experiencing spontaneous abortion.

Integrin heterodimer α9 ß1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state.

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α9 and ß1 subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII-like repeats. Therefore, this study re-evaluated the existence of integrin α9 ß1 actively functioning on the plasma membrane of porcine SSCs using full-length native tenascin C with FNIII-like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α9 ß1 on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α9 ß1 significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α9 and ß1 subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.

Establishment of an electroporation-mediated gene delivery system in porcine spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are a useful tool for the generation of genetically modified transgenic sperm. As a result, the transfer of specific genes into the cytoplasm of SSCs is crucial for the successful generation of transgenic sperm. Here, we report electroporation conditions optimized for SSCs derived from the porcine testis. The highest transfection efficiency and cell viability were observed in porcine SSCs transfected with 1 µg transgenic vector with a single electric pulse from an electroporator at a voltage of 100 V and a capacitor setting of 250 µF. The transfection efficiency and cell viability were constant regardless of the size of the transgenic vector. Furthermore, we did not detect loss of spermatozoa differentiation potential in the transfected porcine SSCs. From these results, we confirm that this electroporation-based gene delivery system can effectively introduce foreign DNA into the genome of porcine SSCs without any loss of the original porcine SSC characteristics, which will be important in the generation of mosaicism-free transgenic pigs produced from transgenic porcine sperm.

Changes of Microbial Diversity During Swine Manure Treatment Process.

We investigated microbial diversity in a manure storage tank (MST) storing untreated manure and an aeration tank (AT) during swine manure treatment process using the next-generation sequencing in order to find the aeration effect on microbial diversity. Proteobacteria were more abundant in the AT group than in the MST group and may include denitrifying bacteria contributing to nitrous oxide (N2O) emission or aerobic bacteria stimulated by oxygen. The opposite held true for the phyla Bacteroidetes and Firmicutes that may include anaerobic bacteria inhibited under aerobic conditions in the AT group.

Integrin Heterodimers Expressed on the Surface of Porcine Spermatogonial Stem Cells.

To date, in vitro culture systems able to sufficiently expand the small population of spermatogonial stem cells (SSCs), a tool for the development of sperm-mediated gene transfer techniques in transgenic pigs, in the porcine seminiferous tubule have not been reported. Therefore, as a step toward engineering a noncellular niche to support the in vitro maintenance of porcine SSC self-renewal, we investigated the types of integrin heterodimers that are expressed and functional on their membrane. The α and ß integrin subunit protein expressions were analyzed using immunocytochemistry and fluorescence immunoassay, and the function of integrin heterodimers was confirmed by attachment and antibody inhibition assays. The integrin subunits, α3, α4, α5, α6, α8, α9, αV, and ß1, were identified on the surface of them. Moreover, they showed significantly increased adhesion to fibronectin, laminin, and vitronectin, and functional blocking of integrin α4ß1, α6ß1, or αVß1 significantly inhibited adhesion to these molecules. They showed significantly decreased adhesion to tenascin C and functional blocking of integrin α5ß1 did not significantly inhibit adhesion to fibronectin. Accordingly, we confirmed that the integrin heterodimers α4ß1, α6ß1, and αVß1 actively function on the surface of undifferentiated porcine SSCs, whereas α3, α5, α8, and α9 are present in inactive forms.

Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

Age-related change of Iba-1 immunoreactivity in the adult and aged gerbil spinal cord.

In the present study, we examined change of ionized calcium-binding adapter molecule 1 (Iba-1) in the adult and aged gerbil spinal cords. Significant change of morphological feature and neuronal cell loss were not observed in both adult and aged spinal cords of gerbil after NeuN immunohistochemistry and Fluoro-Jade B histofluoresce staining. Iba-1-immunoreactive microglia broadly distributed in the spinal cord. Most of Iba-1-immunoreactive microglia showed ramified forms in the adult gerbil cervical and lumbar spinal cords. However, morphological changes of Iba-1-immunoreactive microglia were observed in the cervical and lumbar regions of the aged gerbil spinal cord. These microglia were showed a hypertrophied body with shortened swollen processes which was characteristic of activated microglia. In addition, Iba-1 protein level significantly higher in aged cervical and lumbar spinal cords than those in the adult gerbil. The present study showed an increase of activated forms of Iba-1-immunoreactive microglia and its protein level without marked changes in morphological features and neuronal loss in the aged spinal cord compared to those in the adult gerbil spinal cord. This result suggests that the increase of Iba-1 expression in the aged spinal cord may be closely associated with age-related changes in aged gerbil spinal cord.

Identification of capacitation inducers customized to sperm retrieved from inbred mouse epididymis.

Acquisition of sperm capacitation post-ejaculation into the female reproductive tract is an essential step in the fertilization process. Accordingly, during in vitro fertilization, successful fertilization requires the induction of capacitation in epididymis-retrieved sperm. To date, many candidate substances have been considered as capacitation inducers; however, there are no reports on the efficiency of inducing sperm capacitation among the diverse inducers. Therefore, we attempted to determine the inducer with the best capacitation in inbred mouse sperm by comparing the capacitation performance of a variety of inducers and the percentage of in vitro fertilization-generated zygotes. Calcium chloride, progesterone, bovine serum albumin (BSA), heparin, and lysophosphatidylcholine (Lyso-PC) were used as candidate capacitation inducers. Optimized concentrations of each inducer (2.7 mM calcium, 15 µM progesterone, 0.3% (w/v) BSA, 50 mM heparin, and 100 µM Lyso-PC) were determined based on the ratio of sperm showing an acrosome reaction using Coomassie G-250 blue staining. Calcium at 2.7 mM showed the strongest capacitation induction compared to the other inducers. In vitro fertilization was performed using sperm incubated in each inducer and the ratio of fertilized oocytes was determined. Calcium at 2.7 mM and 0.3% (w/v) BSA showed the highest fertilization rates compared to 15 µM progesterone, 50 mM heparin, and 100 µM Lyso-PC. From these results, we found that 2.7 mM calcium and 0.3% (w/v) BSA were the most effective sperm capacitation inducers of mouse sperm for in vitro fertilization.

Effects of Culture Dimensions on Maintenance of Porcine Inner Cell Mass-Derived Cell Self-Renewal.

Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.

Integrins functioning in uterine endometrial stromal and epithelial cells in estrus.

Here, as a basic study in the construction of a non-cellular niche that supports artificial organization of three-dimensional endometrial tissue, we defined the types of integrin heterodimers that are expressed transcriptionally, translationally and functionally in endometrial stromal (ES) and endometrial epithelial (EE) cells isolated from the mouse uterus in estrus. Gene and protein expression of integrin subunits were analyzed at the transcriptional and translational level by real-time PCR and fluorescent immunoassay, respectively. Moreover, the functionality of integrin heterodimers was confirmed by attachment and antibody inhibition assays. Itga2, Itga5, Itga6, Itga9, Itgav, Itgb1, Itgb3 and Itgb5 in ES cells, and Itga2, Itga5, Itga6, Itga7, Itga9, Itgav, Itgb1, Itgb3, Itgb4, Itgb5 and Itga6 and in EE cells showed significantly higher transcriptional levels than the other integrin subunits. Furthermore, translational expression of the total integrin α and ß subunit genes that showed increased transcription was determined in ES and EE cells. ES cells showed significantly increased adhesion to collagen I, fibronectin and vitronectin, and functional blocking of integrin α2, α5 or αV significantly inhibited adhesion to these molecules. Moreover, EE cells showed significantly increased adhesion to collagen I, fibronectin, laminin and vitronectin, and functional blocking of integrin α2, α5, α6 or αV significantly inhibited adhesion to these molecules. Accordingly, we confirmed that integrin α2ß1, α5ß1, αVß1, αVß3 and/or αVß5, and integrin α2ß1, α5ß1, α6ß1 and/or α6ß4, αVß1, αVß3 and/or αVß5, actively function on the surface of ES and EE cells from mouse uterus in estrus phase, respectively.

Gelatin Directly Enhances Neurogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stem Cells Without Stimulation of Neural Progenitor Cell Proliferation.

Gelatin has been reported to induce generation of mesenchymal stem cells (MSCs) with enhanced potential of differentiation into neuronal lineage cells. However, the presence of various cell types besides MSCs in bone marrow has raised doubts about the effects of gelatin. In the following report, we determined whether gelatin can directly enhance neurogenic differentiation potential in MSCs without proliferation of neural progenitor cells (NPCs). MSCs comprised a high proportion of bone marrow-derived primary cells (BMPCs) and gelatin induced significant increases in MSC proliferation during primary culture, and the proportion of MSCs was maintained at more than 99% throughout the subculture. However, NPCs comprised a low percentage of BMPCs and a decrease in proliferation was detected despite gelatin treatment during the primary culture, and the proportion of subcultured NPCs gradually decreased. In a similar manner, MSCs exposed to gelatin during primary culture showed more enhanced neurogenic differentiation ability than those not exposed to gelatin. Together, these results demonstrate that gelatin directly enhances neurogenic differentiation in bone marrow-derived MSCs without stimulating NPC proliferation.