Abstract
Introduction: In vitro expansion of primary human bone marrow stem cells (hBMSCs) is necessary to obtain sufficient cells for therapeutic uses. Unfortunately, hBMSCs rapidly lose their osteogenic differentiation potential during expansion, significantly limiting their applications. Signal transducer and activator of transcription 4 (STAT4) is known to play roles in cell migration, proliferation, and differentiation. This study aimed to determine the expression and the role of STAT4 during the expansion of hBMSCs. Methods: STAT4 expression in different passages of hBMSCs was evaluated using qRT-PCR and Western blotting. RNA interference and adeno-associated virus serotype 2-mediated gene overexpression were employed to assess the function of STAT4. RNA samples from STAT4-overexpressing hBMSCs were analyzed by RNA-seq to identify differentially expressed genes (DEGs), followed by bioinformatics analyses to determine the pathways affected by STAT4. Results: STAT4 expression progressively decreases during the in vitro expansion of hBMSCs, concomitant with the loss of osteogenic differentiation potential. STAT4 knockdown in early passage hBMSCs significantly inhibits their osteogenic differentiation, evidenced by markedly reduced calcium deposition and downregulation of osteogenic markers. STAT4 knockdown also reduces hBMSCs’ proliferation ability. Conversely, STAT4 overexpression notably increases calcium deposition in passage 3 to passage 7 cells, suggesting that enhanced STAT4 expression can mitigate the loss of osteogenic potential during hBMSC expansion. Transcriptomic analysis revealed DEGs in STAT4-overexpressing hBMSCs. Subsequent bioinformatics analyses indicated that some of these DEGs are involved in pathways regulating cell differentiation and senescence. Conclusion: The in vitro expansion of hBMSCs leads to the downregulation of STAT4, which contributes to the impairment of their osteogenic potential and may affect cell self-renewability. This study provides insight into the molecular mechanisms underlying the loss of osteogenic differentiation during hBMSC expansion and identifies STAT4 as a potential target for hBMSC-based bone regeneration therapies.
Journal Section:
Stem Cells
References
1.
Vidal
L
, Kampleitner
C
, Brennan
MÁ
, Hoornaert
A
, Layrolle
P
. Reconstruction of large skeletal defects: current clinical therapeutic strategies and future directions using 3D printing
. Front Bioeng Biotechnol
. 2020
;8
:61
. 2.
Migliorini
F
, La Padula
G
, Torsiello
E
, Spiezia
F
, Oliva
F
, Maffulli
N
. Strategies for large bone defect reconstruction after trauma, infections or tumour excision: a comprehensive review of the literature
. Eur J Med Res
. 2021
;26
(1
):118
. 3.
Redondo
LM
, García
V
, Peral
B
, Verrier
A
, Becerra
J
, Sánchez
A
, et al. Repair of maxillary cystic bone defects with mesenchymal stem cells seeded on a cross-linked serum scaffold
. J Craniomaxillofac Surg
. 2018
;46
(2
):222
–9
. 4.
Gómez-Barrena
E
, Rosset
P
, Gebhard
F
, Hernigou
P
, Baldini
N
, Rouard
H
, et al. Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial
. Biomaterials
. 2019
;196
:100
–8
. 5.
Oryan
A
, Kamali
A
, Moshiri
A
, Baghaban Eslaminejad
M
. Role of mesenchymal stem cells in bone regenerative medicine: what is the evidence
. Cells Tissues Organs
. 2017
;204
(2
):59
–83
. 6.
Hernigou
P
, Poignard
A
, Beaujean
F
, Rouard
H
. Percutaneous autologous bone-marrow grafting for nonunions: influence of the number and concentration of progenitor cells
. J Bone Joint Surg Am
. 2005
;87
(7
):1430
–7
. 7.
Marolt Presen
D
, Traweger
A
, Gimona
M
, Redl
H
. Mesenchymal stromal cell-based bone regeneration therapies: from cell transplantation and tissue engineering to therapeutic secretomes and extracellular vesicles
. Front Bioeng Biotechnol
. 2019
;7
:352
. 8.
Pittenger
MF
, Mackay
AM
, Beck
SC
, Jaiswal
RK
, Douglas
R
, Mosca
JD
, et al. Multilineage potential of adult human mesenchymal stem cells
. Science
. 1999
;284
(5411
):143
–7
. 9.
Chu
DT
, Phuong
TNT
, Tien
NLB
, Tran
DK
, Thanh
VV
, Quang
TL
, et al. An update on the progress of isolation, culture, storage, and clinical application of human bone marrow mesenchymal stem/stromal cells
. Int J Mol Sci
. 2020
;21
(3
):708
. 10.
Banfi
A
, Muraglia
A
, Dozin
B
, Mastrogiacomo
M
, Cancedda
R
, Quarto
R
. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy
. Exp Hematol
. 2000
;28
(6
):707
–15
. 11.
Chen
J
, Sotome
S
, Wang
J
, Orii
H
, Uemura
T
, Shinomiya
K
. Correlation of in vivo bone formation capability and in vitro differentiation of human bone marrow stromal cells
. J Med Dent Sci
. 2005
;52
(1
):27
–34
. 12.
Fu
X-Y
, Schindler
C
, Improta
T
, Aebersold
R
, Darnell
JE
Jr. The proteins of ISGF-3, the interferon alpha-induced transcriptional activator, define a gene family involved in signal transduction
. Proc Natl Acad Sci U S A
. 1992
;89
(16
):7840
–3
. 13.
Schindler
C
, Fu
X-Y
, Improta
T
, Aebersold
R
, Darnell
JE
Jr. Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha
. Proc Natl Acad Sci U S A
. 1992
;89
(16
):7836
–9
. 14.
Levy
DE
, Darnell
JE
Jr. Stats: transcriptional control and biological impact
. Nat Rev Mol Cell Biol
. 2002
;3
(9
):651
–62
. 15.
Darnell
JE
Jr. STATs and gene regulation
. Science
. 1997
;277
(5332
):1630
–5
. 16.
Awasthi
N
, Liongue
C
, Ward
AC
. STAT proteins: a kaleidoscope of canonical and non-canonical functions in immunity and cancer
. J Hematol Oncol
. 2021
;14
(1
):198
. 17.
Kim
S
, Koga
T
, Isobe
M
, Kern
BE
, Yokochi
T
, Chin
YE
, et al. Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation
. Genes Dev
. 2003
;17
(16
):1979
–91
. 18.
Tajima
K
, Takaishi
H
, Takito
J
, Tohmonda
T
, Yoda
M
, Ota
N
, et al. Inhibition of STAT1 accelerates bone fracture healing
. J Orthop Res
. 2010
;28
(7
):937
–41
. 19.
Wang
M
, Dai
T
, Meng
Q
, Wang
W
, Li
S
. Regulatory effects of miR-28 on osteogenic differentiation of human bone marrow mesenchymal stem cells
. Bioengineered
. 2022
;13
(1
):684
–96
. 20.
Holland
SM
, DeLeo
FR
, Elloumi
HZ
, Hsu
AP
, Uzel
G
, Brodsky
N
, et al. STAT3 mutations in the hyper-IgE syndrome
. N Engl J Med
. 2007
;357
(16
):1608
–19
. 21.
Minegishi
Y
, Saito
M
, Tsuchiya
S
, Tsuge
I
, Takada
H
, Hara
T
, et al. Dominant-negative mutations in the DNA-binding domain of STAT3 cause hyper-IgE syndrome
. Nature
. 2007
;448
(7157
):1058
–62
. 22.
Itoh
S
, Udagawa
N
, Takahashi
N
, Yoshitake
F
, Narita
H
, Ebisu
S
, et al. A critical role for interleukin-6 family-mediated Stat3 activation in osteoblast differentiation and bone formation
. Bone
. 2006
;39
(3
):505
–12
. 23.
Yadav
PS
, Feng
S
, Cong
Q
, Kim
H
, Liu
Y
, Yang
Y
. Stat3 loss in mesenchymal progenitors causes Job syndrome-like skeletal defects by reducing Wnt/β-catenin signaling
. Proc Natl Acad Sci U S A
. 2021
;118
(26
):e2020100118
. 24.
Zhou
S
, Dai
Q
, Huang
X
, Jin
A
, Yang
Y
, Gong
X
, et al. STAT3 is critical for skeletal development and bone homeostasis by regulating osteogenesis
. Nat Commun
. 2021
;12
(1
):6891
. 25.
Teglund
S
, McKay
C
, Schuetz
E
, Van Deursen
JM
, Stravopodis
D
, Wang
D
, et al. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses
. Cell
. 1998
;93
(5
):841
–50
. 26.
Lee
KM
, Park
KH
, Hwang
JS
, Lee
M
, Yoon
DS
, Ryu
HA
, et al. Inhibition of STAT5A promotes osteogenesis by DLX5 regulation
. Cell Death Dis
. 2018
;9
(11
):1136
. 27.
Joung
YH
, Lim
EJ
, Darvin
P
, Chung
SC
, Jang
JW
, Do Park
K
, et al. MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs
. PLoS One
. 2012
;7
(10
):e47477
. 28.
Kaplan
MH
, Sun
Y-L
, Hoey
T
, Grusby
MJ
. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice
. Nature
. 1996
;382
(6587
):174
–7
. 29.
Thierfelder
WE
, van Deursen
JM
, Yamamoto
K
, Tripp
RA
, Sarawar
SR
, Carson
RT
, et al. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells
. Nature
. 1996
;382
(6587
):171
–4
. 30.
Chang
HC
, Han
L
, Goswami
R
, Nguyen
ET
, Pelloso
D
, Robertson
MJ
, et al. Impaired development of human Th1 cells in patients with deficient expression of STAT4
. Blood
. 2009
;113
(23
):5887
–90
. 31.
Rong
W
, Rome
CP
, Dietrich
MA
, Yao
S
. Decreased CRISPLD2 expression impairs osteogenic differentiation of human mesenchymal stem cells during in vitro expansion
. J Cell Physiol
. 2023
;238
(6
):1368
–80
. 32.
Aurnhammer
C
, Haase
M
, Muether
N
, Hausl
M
, Rauschhuber
C
, Huber
I
, et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences
. Hum Gene Ther Methods
. 2012
;23
(1
):18
–28
. 33.
Gaj
T
, Schaffer
DV
. Adeno-associated virus–mediated delivery of CRISPR–Cas systems for genome engineering in mammalian cells
. Cold Spring Harb Protoc
. 2016
;2016
(11
):pdb.prot086868
. 34.
Yao
S
, Rong
W
, Yuan
Y
. Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs)
. Stem Cell Investig
. 2023
;10
(3
). 35.
Sherman
BT
, Hao
M
, Qiu
J
, Jiao
X
, Baseler
MW
, Lane
HC
, et al. DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update)
. Nucleic Acids Res
. 2022
;50
(W1
):W216
–21
. 36.
Huang
DW
, Sherman
BT
, Lempicki
RA
. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources
. Nat Protoc
. 2009
;4
(1
):44
–57
. 37.
Chen
D
, Gong
Y
, Xu
L
, Zhou
M
, Li
J
, Song
J
. Bidirectional regulation of osteogenic differentiation by the FOXO subfamily of Forkhead transcription factors in mammalian MSCs
. Cell Prolif
. 2019
;52
(2
):e12540
. 38.
Sanpaolo
ER
, Rotondo
C
, Cici
D
, Corrado
A
, Cantatore
FP
. JAK/STAT pathway and molecular mechanism in bone remodeling
. Mol Biol Rep
. 2020
;47
(11
):9087
–96
. 39.
Damerau
A
, Gaber
T
, Ohrndorf
S
, Hoff
P
. JAK/STAT activation: a general mechanism for bone development, homeostasis, and regeneration
. Int J Mol Sci
. 2020
;21
(23
):9004
. 40.
Minegishi
Y
, Saito
M
, Morio
T
, Watanabe
K
, Agematsu
K
, Tsuchiya
S
, et al. Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired immunity
. Immunity
. 2006
;25
(5
):745
–55
. 41.
Hu
X
, Li
J
, Fu
M
, Zhao
X
, Wang
W
. The JAK/STAT signaling pathway: from bench to clinic
. Signal Transduct Target Ther
. 2021
;6
(1
):402
. 42.
Teng
Y
, Xu
J
, Wang
Y
, Wen
N
, Ye
H
, Li
B
. Combining a glycolysis-related prognostic model based on scRNA-Seq with experimental verification identifies ZFP41 as a potential prognostic biomarker for HCC
. Mol Med Rep
. 2024
;29
(5
):78
–15
. 43.
Meier-Stiegen
F
, Schwanbeck
R
, Bernoth
K
, Martini
S
, Hieronymus
T
, Ruau
D
, et al. Activated Notch1 target genes during embryonic cell differentiation depend on the cellular context and include lineage determinants and inhibitors
. PLoS One
. 2010
;5
(7
):e11481
. 44.
Fiddes
IT
, Lodewijk
GA
, Mooring
M
, Bosworth
CM
, Ewing
AD
, Mantalas
GL
, et al. Human-specific NOTCH2NL genes affect notch signaling and cortical neurogenesis
. Cell
. 2018
;173
(6
):1356
–69.e22
. 45.
Lin
SJ
, Zhang
Y
, Liu
NC
, Yang
DR
, Li
G
, Chang
C
. Minireview: pathophysiological roles of the TR4 nuclear receptor: lessons learned from mice lacking TR4
. Mol Endocrinol
. 2014
;28
(6
):805
–21
. 46.
Lin
S-J
, Ho
H-C
, Lee
Y-F
, Liu
N-C
, Liu
S
, Li
G
, et al. Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis
. Reprod Biol Endocrinol
. 2012
;10
:43
–7
. © 2025 S. Karger AG, Basel
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
2025
You do not currently have access to this content.
