Shinshu Med J, 72⑷：227～237, 2024

Unraveling the Influence of Folic Acid on Osteogenic Differentiation :Insights from Induced Pluripotent Stem Cells
Yali Men1）＃, Fengming Yue1）2）＃, Kensuke Kai1）
Chuang Ma2）3）, Daihachiro Tomotsune1）2） and Kohei Johkura1）＊
1） Department of Histology and Embryology, Shinshu University School of Medicine
2） Shinshu University Interdisciplinary Cluster for Cutting Edge Research, Institute for Biomedical Sciences
3） Biomedical Engineering Division, Graduate School of Medicine, Science and Technology, Shinshu University

Folic acid, a crucial B vitamin with established roles in various physiological processes, has gained recognition
for its association with neural and cardiovascular health, cancer prevention, and metabolic syndrome. Despite
these multifaceted roles, its specific influence on bone health, especially during fetal development, remains underexplored. This study employed induced pluripotent stem cells (iPSCs) of human to simulate in vivo osteogenic
differentiation, comprehensively investigating the influence of folic acid on bone formation. As a result, a substantial increase in calcium deposition was revealed by addition of folic acid into the osteogenic differentiation of
iPSCs, indicating an augmented capacity for mineralized tissue formation. The expression of key osteogenic genes(RUNX2, COL1A1, Osteocalcin, PHEX) consistently showed upregulation across various stages of osteogenic
differentiation. Additionally, temporal effects of folic acid were observed, emphasizing its positive impact during
the early preosteoblast stage. The role of folic acid in promoting the differentiation of preosteoblasts into osteoblasts was further confirmed using MC3T3-E1 cells. These findings provide novel insights into the molecularmechanisms underlying the influence of folic acid on bone formation, highlighting the specific stages of osteogenic differentiation where its effects are most pronounced. Shinshu Med J 72 : 227 ―237, 2024
(Received for publication March 29, 2024 ; accepted in revised form April 9, 2024)
Key words : induced pluripotent stem cells (iPSCs), folic acid, osteogenic differentiation, temporal effects

on the broader implications of folic acid, including neu-

Ⅰ Introduction

ral, reproductive, and cardiovascular health, cancer

Folic acid, a vital member of the B-vitamin (B9),prevention, metabolic syndrome, and inflammation7）-9）.

plays a crucial role in physiological processes, includ-

However, extensive studies on folic acid’s specific im-

ing nucleic acid synthesis, amino acid metabolism, andpact on bone health and development are lacking. In

DNA methylation1）2）. The conversion of homocysteine

adults, insufficient folic acid intake has been associatedto methionine is facilitated by folic acid, which is fun-with bone-related issues, including osteoporosis and3）4）. Tra-

heightened osteoclast activity, which may compromise

ditionally known for preventing neural tube defects inbone integrity10）. During pregnancy, folic acid defi-

pregnancy5）6）, contemporary research has shed lightciency is associated not only with neural tube defects

damental for numerous biochemical reactions

but also with potential effects on fetal bone develop＊

Corresponding author : Kohei Johkura

ment11）. Hossein et al. reported that pregnant women

Department of Histology and Embryology,

receiving daily folic acid supplementation (1 mg) until

Shinshu University School of Medicine,
3-1-1 Asahi, Matsumoto, Nagano 390-8621 Japan
E-mail : kohei@shinshu-u.ac.jp＃

These authors contributed equally : Yali Men & Fengming Yue

No. 4, 2024

childbirth exhibited significantly higher plasma levelsof osteoprotegerin (OPG) concentration and lower
soluble receptor activator of nuclear factor-kappa B
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Men・Yue・Kai et al.

ligand (sRANKL) concentrations than those supple­
12）

eages using a tailored protocol that mimicked themented only up to the second trimester . Despite

early stages of bone formation18）19）. This process in­these associations, the precise mechanisms by which

volves the application of specific differentiation fac­

folic acid influences bone formation, during fetal de­tors. Briefly, 2×105 iPSCs were maintained undervelopment, remain intensely investigated.

feeder-free conditions. Differentiation was initiated in

To address this issue, we utilized induced pluripo­

gelatin-coated 24-well plates using a mixture of 20 ％tent stem cells (iPSCs) of human, a groundbreaking

mTeSR1 medium (StemCell Technologies, Vancou­

discovery by Yamanaka et al.

13）
14）

. iPSCs possess thever, BC, CA) and 80 ％ osteogenic differentiation me­

unique ability to be reprogrammed from adult cells,

dium. The osteogenic differentiation medium com­offering an ethically clear approach to studying hu­

prised KnockOut DMEM (Gibco, Grand Island, NY,

man development, disease modeling, and regenerativeUSA), supplemented with 20 ％ FBS (Gibco), 1 ％ non-medicine. This innovation allows modeling embryonicessential amino acids (Wako), 0.1 mM 2-mercaptoeth­

development in a dish, offering a unique vantage point

anol (Sigma), 2 mM Gluta-MAX (Wako), 10 mM glycer­

to study gene and drug effects at each developmental

ol-2-phosphate (Sigma), 1 nM dexamethasone (Sigma),stage15）-17）.

50 μg/mL L-ascorbic acid 2-phosphate sesquimagne­

The primary objective of this study was to investi­

sium salt hydrate (Sigma), 1 μM all-trans-retinoic acidgate osteogenesis using iPSCs and scrutinize the ef­(Sigma), and 10 μM rock inhibitor Y-27632 (Wako). On

fects of folic acid supplementation, elucidating the com­day 2, the medium was replaced with 100 ％ osteo­

plex relationship between folic acid and bone formation,

genic differentiation medium, excluding Y-27632, andwith a particular focus on understanding how folic

subsequently changed every two days. During theacid influences the molecular and cellular mecha­

induction of differentiation, we supplemented the cul­

nisms involved in bone development across different

ture medium with 50 μM folic acid (Wako, Osaka,stages. This investigation holds the potential to pro­Japan) to investigate the impact of folic acid on bonevide valuable insights into therapeutic approaches

development. This concentration of folic acid was de­for enhancing bone health and preventing associatedtermined based on our preliminary experiment ofconditions in both adults and developing fetuses.

osteogenic differentiation by using a gradient of 0,Ⅱ
ＡMaterials and Methods

10, 25, 50, 100, 150, and 200 μM.
ＣOsteogenic differentiation of MC3T3-E1
Differentiation was induced using a medium con­

Cell culture and maintenance of iPSCs
Human iPSC line 253G1 (Riken Brc, Tsukuba, Japan)

taining 50 μg/ml vitamin C (Wako) and 10 mM β-

was utilized in this study. iPSC culture involved feeder

glycerophosphate (Wako). To further investigate thelayers of inactivated mouse embryonic fibroblasts

factors influencing osteoblast differentiation, an addi­(MEFs) in KnockOut

TM

DMEM/F-12 (Gibco), supple­

tional component, folic acid (Wako), was added to themented with 20 ％ knockout-serum replacement (Gib­

culture medium at the concentration of 10 μM or 50co), 100 mM non-essential amino acids (Wako, Japan),μM.

1 mM sodium pyruvate (Wako), 100 mM 2-mercap­

Ｄ

Alizarin staining for calcium deposition

toethanol (Sigma-Aldrich, St. Louis, MO, USA), 50 U/

For Alizarin Staining cells were initially fixed in amL penicillin, and 50 mg/mL streptomycin (Wako).

culture dish using 4 ％ paraformaldehyde (PFA, Wako).

The murine calvaria-derived osteoblast-like cell line

After fixation, 1 ％ Alizarin staining solution (Muto(MC3T3-E1, Riken BRC, Ibaraki, Japan）cells were

Chemicals, Tokyo, Japan) was applied and incubatedcultured in α-MEM medium supplemented with 10 ％at room temperature for 10 min to promote the Aliz­

FBS.

arin staining reaction. Subsequently, the cells wereＢ

washed with water to remove the excess staining

Osteogenic differentiation of iPSCs
The iPSCs were differentiated into osteogenic lin­228

solution. Microscopic observations were conducted to
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Folic acid’s impact on osteogenic differentiation : iPSC insights

visualize alizarin-stained (red crystalline deposits)

antibodies at 4 ℃ overnight. Antibodies used for im­

cells.

munohistochemistry were against osteocalcin (1 :Ｅ

100 ; Thermo Fisher Scientific) and Collagen I (1 :Quantification of calcium deposition
iPSCs-derived osteogenic cells were exposed to 1

100 ; Santa Cruz Biotechnology). Antibody specificity

N hydrochloric acid (HCl) for 24 h. Calcium content inwas confirmed by validation using appropriate tis­the HCl supernatant was quantified using an o-cresolsues or cells as positive controls. Antigen localization

phthalein complexone (OCPC) kit (N-assay L Ca ;was visualized using secondary antibodies conjugatedNittobo Medical, Tokyo, Japan) at 660 nm (referencewith fluorescein, specifically, anti-rabbit or anti-mouse

wavelength : 800 nm) with a microplate spectropho­

immunoglobulin G (IgG) (Alexa Fluor 568 and 488 ; Mo­

tometer. Calcium content was normalized to the cell

lecular Probes Inc., Eugene, OR, USA). Imaging was

number.

performed using confocal laser scanning microscopy

Ｆ(Leica TCS SP8) and fluorescence microscopy (Keyence

Real-time RT-PCR analysis
Total cellular RNA was extracted using the TRIzol

gure 1

reagent (Invitrogen, Carlsbad, CA, USA) following

BZ-X800).
Ｈ

Statistical analysis

the manufacturer’s instructions. Genomic DNA was

Data from three independent experiments are ex­removed by DNase treatment using a PrimeScript

pressed as mean±standard deviation. Statistical sig­

RT reagent kit with a gDNA eraser (Takara Bio,nificance for qRT-PCR and other assays was assessed

Shiga, Japan). Subsequently, 1 μg of total RNA wasusing unpaired two-tailed Student’s t-tests, with areverse-transcribed into single-stranded DNA using

p-value＜0.05 considered significant.

random primers and reverse transcriptase. qRT-PCR

Ⅲ

analysis was conducted as described previously20）
using a Thermal Cycler Dice Real-Time System (Ta­Ａ

Results

Osteogenic differentiation from iPSCs

kara Bio). mRNA levels were normalized to beta-

To explore the effect of folic acid on osteogenic

actin, and the original cycle threshold values weredifferentiation, iPSCs with self-renewal and pluripo­

analyzed using the 2-ΔΔCt method.

tent differentiation capabilities were used. Sequential

Ｇ

iPSC differentiation mimicked the osteogenic lineage,

Immunostaining for protein expression
Cells were fixed in 4 ％ paraformaldehyde for 10

which was induced to simulate in vivo bone formation,

min, washed with 0.01 M phosphate-buffered saline.

encompassing osteoprogenitor, pre-osteoblast, osteo­

Subsequently, the cells were incubated with primary

blasts, and osteocytes (Fig. 1).

Fig. 1 Osteogenic differentiation from iPSCs. The iPSCs were differentiated into osteogenic lineages using a tailored protocolthat mimicked the early stages of bone formation, including osteoprogenitor, pre-osteoblast, osteoblasts, and osteocytes.
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Fig. 2 Impact of folic acid on osteogenic differentiation and calcium deposition in iPSCs. (A) Representative images of
Alizarin Red staining (red granules) during osteogenic differentiation in the folic acid-added group and the untreat­ed group (control). (B, C) The evaluation of bone formation on the indicated day was quantified with a dissolved
alizarin solution (B) and the deposition of calcium (C). FA : folic acid. The results were normalized by cell number.
Data expressed as the mean±SD (n＝3) ; ＊p＜0.05, ＊＊p＜0.01.

Ｂ

Calcium deposition in bone-like nodules

differentiation on days 4, 7, and 10 than the control

Alizarin Red staining was used to visualize andgroup (Fig. 2B), and calcium quantification using

quantify calcium deposition in the bone-like nodules,

OCPC showed a significant increase in calcium depo­

which is a crucial indicator of osteoblast activity. Aliz­sition in the folic acid-supplemented group on daysarin Red staining on days 2, 4, 7, and 10 revealed a4, 7, and 10 (Fig. 2C). These results suggested that

noticeable trend of darker staining in the folic acid-folic acid supplementation promoted calcium deposi­

supplemented experimental group compared to thetion during osteogenic differentiation.

control group, particularly on days 7 and 10 (Fig. 2A).

Ｃ Gene and protein expression profiling at

In addition, a bone-like nodule dissolution experiment

different stages of osteogenic differentiation

was conducted by adding acetic acid to dissolve pig­

Real-time RT-PCR analysis was employed to as­

ments. Absorbance measurements of the dissolved

sess the expression levels of genes related to osteo­

solution were conducted to assess calcium phosphate

genic differentiation, including RUNX2, COL1A1, Os-

content, indicative of osteoblast and osteocyte differ­teocalcin (OCN), and PHEX (Fig. 3A). This molecular

entiation. The higher Alizarin OD in the folic acid-approach provided valuable data on the influence of

supplemented group exhibited greater osteoblast

folic acid on the genetic regulation of bone formation.

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Folic acid’s impact on osteogenic differentiation : iPSC insights

Fig. 3 Gene and protein expression profiling during osteogenic differentiation. (A) Quantification of the expressionof osteogenic differentiation stage-related genes RUNX2, COL1A1, OCN, and PHEX. Data expressed as
the mean±SD (n＝3) ; ＊p＜0.05, ＊＊p＜0.01. (B) Immunostaining of type I collagen (COL1, red), osteoblast-spe­cific protein Osteocalcin (OCN, green), and Phalloidin (red). The presence of the cells was confirmed bystaining nuclei with DAPI (blue). Scale bars : 30 μm. FA : folic acid

RUNX2, a scaffold for nucleic acids and regulatorylagen, the fibrillar collagen present in various con­factors involved in skeletal gene expression, exhibit­

nective tissues, including bone and cartilage, exhibit­

ed significant differences on days 4 and 7, indicating

ed elevated expression in the folic acid-supplemented

that folic acid promotes bone formation. COL1A1,

group on days 2, 4, and 7. OCN, which is among thewhich encodes the principal component of type I col­most abundant proteins in bone and is produced ex­

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clusively by osteoblasts, showed maximum expres­larger and darker bone-like nodules compared to thesion in the folic acid-supplemented group on day 7,other groups (Fig. 4A). Key genes associated with

with a significant difference compared to the control.

different stages of bone development were selected,

PHEX, a marker of osteocytes, was highly expressed

and their expression was analyzed (Fig. 4B). On day

in the folic acid-supplemented group on day 10. These

1, the expression of the osteoprogenitor-related gene

findings suggested that folic acid influences gene ex­

RUNX2 showed a significant increase in the folic acid-pression at various stages of osteogenic differentia­

supplemented group. On day 2, an increase in thetion, supporting its potential role in promoting bone

expression of the pre-osteoblast-related gene ALP

formation.

was observed although statistical analysis did not re­

Immunostaining was employed to detect and visu­

veal a significant difference. On day 4, the expression

alize specific proteins linked to osteoblasts and osteo­of the osteoblast-related gene Bone sialoprotein (BSP)

cytes, including Type I collagen (COL1) and osteocal­

was significantly increased in the group that receivedcin (OCN) (Fig. 3B). Osteoblasts synthesize bone matrix

folic acid on the 2nd day of differentiation. On day 7,

via collagen secretion and hydroxyapatite crystal

the expression of the osteoblast-related genes OCN

deposition. COL1 expression in bone-like nodules in­and COL1A1 showed a significant increase in the

duced from iPSCs showed a uniform distribution ingroup that received folic acid on day0 and day2. On

the control and folic acid-supplemented group. Nota­

day 10, the expression of the osteocyte-related gene

bly, a more pronounced positive signal was discerned

PHEX significantly increased in all folic acid-supple­

in the folic acid group on day 7. OCN, exclusively pro­mented groups, except for that supplemented on day 7,

duced by osteoblasts, serves as a standard osteoblast

with the most notable increase observed in the group

marker. On the fourth day of induction, both the con­that received folic acid on the 2nd day. These results

trol and folic acid-supplemented groups exhibited

indicate that the addition of folic acid has affected

OCN-positive cells within the bone-like nodules, withdifferent stages of bone development, with the mosta stronger positive signal observed in the folic acid-significant promotion of osteoblast differentiation

added group on days 7 and 10. Following the isolation

observed when folic acid was added during pre-of cells from bone-like nodules through collagenase

osteoblast differentiation.

treatment, staining with phalloidin was conducted toＥ Confirmation of folic acid role in osteoblast

visualize the actin filaments. Most cells isolated on

differentiation using MC3T3-E1 cell line

days 4 and 7 exhibited a columnar morphology con­

To further validate the role of folic acid in osteo­sistent with osteoblasts. As mineralization progress­

blast differentiation, the MC3T3-E1 cell line was em­

es, they transform into dendritic-shaped cells withployed, and cells were treated with 10 μM and 50 μM

spines on day 10 (arrowhead in Fig. 3B). As osteo­

folic acid. On days 5th and 10th day of differentiation,

genic differentiation, osteoblasts gradually transform

Alizarin Red staining revealed a deeper red color in

into mature osteocytes, characterized by morphologi­

the two folic acid-added groups (Fig. 5A). Gene ex­cal changes such as a reduction in cell volume andpression analysis of COL1A2 and OCN showed sig­

the adoption of a star-shaped appearance (arrow innificant enhancement in the folic acid supplemented

Fig. 3B).

groups (Fig. 5B). Additionally, on day 10 of differen­

Ｄ Folic acid effects at different stages oftiation induction, cells were passage into single cells

osteogenic development

and subjected to immunostaining. The results showed

To investigate the role of folic acid at various stag­an increased number of OCN-positive cells and en­es of bone development, folic acid was added on days

hanced expression of COL1 protein in the folic acid-0, 2, 4, and 7 during the induction of differentiation.

supplemented groups. Phalloidin staining further

Visual observation of alizarin staining revealed that

demonstrated that a greater proportion of cells in thethe group treated with folic acid on day 2 exhibited

folic acid-supplemented group underwent a transition

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Folic acid’s impact on osteogenic differentiation : iPSC insights

Fig. 4 Folic acid effects at various stages of bone development. (A) Images of Alizarin Red staining on Day 10 in thecontrol and folic acid-added group. Folic acid was added on Day 0, 2, 4, and 7. Note the larger bone-like nodules
with deep red staining at the upper left part of the image of addition on Day 2 (D2(＋)). FA : folic acid. (B) Key
gene expression at different stages of osteogenic differentiation, RUNX2, ALP, BSP, COL1A1, OCN, and
PHEX, expressed as a value relative to that of the control group on Day 0. Data expressed as the mean±SD(n＝3) ; ＊p＜0.05, ＊＊p＜0.01.
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Fig. 5 Role of folic acid in osteoblast differentiation using the MC3T3-E1 cell line. (A) Alizarin Red staining on the
5th and 10th days of differentiation for groups treated with varying concentrations of folic acid. (B) Gene ex­pression levels of crucial markers, COL1A2 and OCN. Data expressed as the mean±SD (n＝3) ; ＊＊p＜0.01.
(C) Immunostaining of osteoblast-specific protein Osteocalcin (OCN, green), type I collagen (COL1, red) andPhalloidin (red) on Day 10. Nuclei were counterstained with DAPI (blue). Scale bars : 30 μm. FA : folic acid234

Shinshu Med J. 72

Folic acid’s impact on osteogenic differentiation : iPSC insightsfrom a rectangular to a polygonal shape (Fig. 5C).

deposition, a crucial aspect of bone mineralization.

Collectively, these results support the notion that

Nutrition plays a pivotal role in influencing thefolic acid plays an important role in enhancing osteo­

molecular processes underlying bone health. Multiple

genic differentiation and calcium deposition, particu­

studies22）23） have investigated the regulatory influ­

larly during pre-osteoblast differentiation. These

ence of folic acid on DNA methylation patterns with­findings provide valuable insight into the complex

in osteoblasts, suggesting potential epigenetic mecha­

relationship between folic acid and bone formation.

nisms capable of modulating bone formation. Our

Ⅳ Discussion

study demonstrated the upregulation of crucial osteo­
genic differentiation genes (RUNX2, COL1A1, OCN,

Our study utilized iPSCs to mimic the in vivo osteo­

and PHEX) in the presence of folic acid. Immuno­

genic differentiation process and conducted a com­staining for Type I collagen and osteocalcin further

prehensive investigation into the role of folic acid atprovided visual evidence of enhanced protein expres­various stages of bone formation. Folic acid supple­

sion in the folic acid-supplemented group. Especially,

mentation significantly enhanced calcium deposition

the morphological changes observed during osteogenic

in bone-like nodules, indicating increased osteogenic

differentiation revealed the dynamic role of folic acidactivity. Crucial osteogenic differentiation genes, suchin driving the progression from osteoblasts to mature

as RUNX2, COL1A1, OCN, and PHEX, consistently

osteocytes.

exhibited upregulated in the presence of folic acid.

Considering the age-related changes in bone phys­

Additionally, temporal variations in the effects ofiology, temporal variations in folic acid effects at dis­

folic acid at distinct stages of osteogenic differentia­

tinct stages of osteogenic differentiation were observed.

tion were observed, indicating a time-dependent man­

The darker bone-like nodules and upregulated expres­ner of the responses of osteogenic cells to folic acidsion of osteogenic genes in the group that received

during bone development. Furthermore, the inclusion

folic acid during pre-osteoblast differentiation sug­of the MC3T3-E1 cell line results not only validated

gested the influence on osteoblast differentiation.

our findings with iPSCs but also strengthen the folic

Moon et al.24） reported that early treatment of mes­

acid’s positive influence on osteoblast differentiation.

enchymal stem cells with folic acid significantly pro­This study represents the first in-depth exploration

motes chondrogenic differentiation, emphasizing theof the specific effect of folic acid on the early stages

importance of timing in the supplementation. Throughof bone development.

additional validation experiments on the osteoblast-

Adequate nutrient intake, including folic acid, haslike cell line (MC3T3-E1), we confirmed the crucial

been associated with improved bone mineralization,role of folic acid in osteoblast differentiation, particu­

emphasizing the multifaceted role of nutrition in sup­larly during the transition from pre-osteoblasts toporting skeletal integrity, especially in aging popula­mature osteoblasts. This temporal dimension offers ations. A recent study by Zheng et al.21） demonstrated

dynamic understanding of the effects of folic acid on

a positive correlation between folic acid intake andosteogenic differentiation at different stages. Ourbone mineral density in postmenopausal women. In

study hints at the time-dependent and complex re­

this study, the observed increase in Alizarin Red

sponses of osteogenic cells to folic acid supplementa­

staining and calcium quantification in the folic acid-tion during different phases of bone development.

supplemented group suggested a stimulatory effect

In the context of nutrition and aging, our study

on osteoblast activity. The dissolution experiment

prompts a discussion on the potential significance offurther substantiated these findings, indicating in­folic acid supplementation for pregnant women andcreased calcium phosphate content in the folic acid-older individuals. Future studies could explore thesupplemented group. These results collectively point

optimal dosage and duration of folic acid supplemen­

towards a promoting role of folic acid in calcium

tation for bone health maintenance. Moreover, it isNo. 4, 2024

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Men・Yue・Kai et al.

essential to note that bone development is a complex

lighting that supplementation with folic acid during

process involving various nutrients. Previous stud­pregnancy not only crucially prevents neural tube

ies

25）
26）

have emphasized how folic acid, in conjunc­

defects in the developing fetus but also holds poten­tion with vitamins D, B6, and B12, can synergisticallytial relevance for the skeletal system development.

promote bone health. Understanding these interac­

Further research is needed to elucidate the complex­

tions is essential for developing straightforward di­ities of nutrient interactions and the precise molecular

etary strategies to enhance overall bone formation.

pathways through which folic acid exerts its effects

In conclusion, utilization of iPSCs for in vivo osteo­
genic differentiation provides comprehensive evidenceon osteogenic differentiation, as well as to explore its
therapeutic applications in bone health.

that folic acid plays a pivotal role in enhancing osteo­

Acknowledgments

genic differentiation and calcium deposition, particu­
larly during pre-osteoblast differentiation. These

We are grateful to the Division of Instrumental Re­

findings contribute valuable insights into the molecu­

search, Shinshu University Research Center for Ad­

lar and cellular mechanisms underlying the relation­

vanced Science and Technology.

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