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Article

Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells

by
Yixuan Amy Pei
1,2,
Jhanvee Patel
1 and
Ming Pei
1,3,*
1
Stem Cell and Tissue Engineering Laboratory, Department of Orthopaedics, West Virginia University, Morgantown, WV 26506, USA
2
Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
3
WVU Cancer Institute, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506, USA
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(5), 1932; https://doi.org/10.3390/app14051932
Submission received: 13 December 2023 / Revised: 29 January 2024 / Accepted: 24 February 2024 / Published: 27 February 2024
(This article belongs to the Section Applied Biosciences and Bioengineering)
Browse Figures
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Abstract

:
Adult mesenchymal stem cells (MSCs) are a promising cell source for tissue regeneration. However, ex vivo expansion results in cell senescence; cells lose their proliferation and differentiation capacity. Fetal MSCs can offer an alternative due to their robust proliferation and differentiation capacities, as well as their immune privilege properties. Given the rejuvenation effect of the decellularized extracellular matrix (dECM) on adult MSCs, it remains unknown whether dECM influences the regenerative potential of fetal stem cells. In this study, passage five fetal nucleus pulposus cells (fNPCs) and fetal synovium-derived stem cells (fSDSCs) were expanded on dECMs deposited by fNPCs (NECM) and fSDSCs (SECM) for one passage, with expansion on tissue culture plastic (Plastic) as a control. We found that dECM-expanded fNPCs and fSDSCs exhibited both similarities and differences in the expression of stemness genes and surface markers. Expanded fNPCs yielded more differentiated pellets after chondrogenic induction but exhibited no adipogenic differentiation following adipogenic induction in both the Plastic and dECM groups than the corresponding fSDSC group. Despite a significant increase in fNPCs, the dECM-expanded fSDSCs exhibited no increase in chondrogenic potential; however, compared to the Plastic group, dECM-expanded fSDSCs exhibited a small increase in osteogenic potential and a great increase in adipogenic potential. These results suggest that fNPCs are more sensitive to NECM rejuvenation for cartilage tissue engineering and regeneration; in contrast, the dECMs exhibited limited effects on fSDSC rejuvenation in a chondrogenic capacity, except for enhanced adipogenic capacity following expansion on SECM.

1. Introduction

Adult mesenchymal stem cells (MSCs) are a promising cell source for tissue engineering and regeneration [1]. However, MSCs experience cell senescence in clinical treatment, due to either donor age and/or ex vivo expansion [2,3]. Compared to adult MSCs with median levels of human leukocyte antigen (HLA) class I and low levels of HLA class II, fetal MSCs have low levels of HLA class I and do not express HLA class II, indicating that fetal MSCs have greater immunomodulatory properties [4,5,6]. Moreover, fetal MSCs possess greater proliferation and differentiation capacities compared to adult MSCs, indicating that fetal MSCs may be advantageous for MSC-based tissue regeneration [7,8,9,10].
Despite adult MSCs being prone to aging during ex vivo expansion, the decellularized extracellular matrix (dECM) deposited by MSCs provides a solution to rejuvenating both proliferation and chondrogenic capacity in adult MSCs [11,12]. There is an abundance of research supporting the use of cell-derived dECMs to maintain stemness, prevent cell senescence, and limit oxidative stress when expanding stem cells [13,14,15,16,17,18]. Previously, we found that human adult synovium-derived MSCs (aSDSCs) were rejuvenated after expansion on dECM deposited by fetal SDSCs (fSDSCs), compared to that deposited by aSDSCs [19]. Furthermore, high-passage (senescent) infrapatellar fat pad-derived MSCs (IPFSCs) grown on dECM deposited by low-passage (young) IPFSCs exhibited greater proliferative and chondrogenic capacity, whereas high-passage IPFSCs expanded on dECM deposited by high-passage IPFSCs lost their proliferative and chondrogenic potential [20], indicating that the matrix microenvironment can greatly influence adult MSC regenerative potential. This evidence could also suggest that dECMs deposited by younger, more proliferative cells could have a greater ability to rejuvenate [19,20].
However, it remains unknown whether a fetal matrix microenvironment can influence the regenerative potential of fetal stem cells. In this study, a tissue-specific stem cell for chondrogenesis, SDSCs [21], and a chondrogenic progenitor cell, nucleus pulposus cells (NPCs) [22], were used to characterize fetal MSCs in terms of the expression of stemness genes and surface markers and proliferation capacity, as well as the chondrogenic, adipogenic, and osteogenic potential following expansion on dECMs deposited by fSDSCs (SECM) or fetal NPCs (fNPCs) (NECM). Major matrix proteins were also assessed for expression in dECMs that were deposited by fSDSCs and fNPCs, as well as in expanded cells and multi-differentiated fetal MSCs.

2. Materials & Methods

2.1. Human fNPC and fSDSC Culture

Human fNPCs and fSDSCs [23] (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in complete medium (α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1× PSF (100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL fungizone) (Thermo Fisher Scientific, Waltham, MA, USA)) in a 5% CO2 incubator at 37 °C.

2.2. Preparation of dECMs

Tissue culture plastic (Plastic) flasks were pre-coated with 0.2% gelatin from bovine skin, type B (Millipore Sigma, St. Louis, MO, USA), at 37 °C for 1 h, followed by treatment with 1% glutaraldehyde solution (Thermo Fisher Scientific) and 1 M ethanolamine (Millipore Sigma) for 30 min each. Passage four (P4) fSDSCs and fNPCs were seeded on pre-coated Plastic flasks (3800 cells/cm2). Cells at confluency were treated with 250 μM L-ascorbic acid phosphate (Wako Chemicals USA Inc., Richmond, VA, USA) for an additional 10 days. Afterward, the cells were incubated with a lysis buffer containing 0.5% Triton X-100 (Millipore Sigma) and 20 mM ammonium hydroxide (Millipore Sigma) at 37 °C for 10 min. After cell removal, the dECMs were kept in phosphate buffered solution (PBS)-diluted lysis buffer (1։1) at 4 °C overnight. The next day, the dECMs were washed and stored with PBS containing 1× PSF at 4 °C until use.

2.3. Evaluation of Proliferation, Surface Markers, and Stemness Genes of Expanded fSDSCs and fNPCs

P5 fSDSCs and P5 fNPCs were expanded on Plastic and dECMs deposited by fSDSCs and fNPCs, respectively, for one passage, subsequently followed by the analyses below.
The expanded cells were assessed for cell proliferation using a nucleoside analog Click-iT EdU Alexa Flour™ 647 flow cytometry assay kit (Invitrogen, Eugene, OR, USA). After the expanded cells reached 45% confluence, EdU was added to the cell culture medium at a final concentration of 10 μM. After 20 h of incubation, the harvested cells (3 × 105 in each group) were incubated with Click-iT™ reaction cocktail (1×) in the dark for 30 min. Fluorescence was detected by a BD FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, USA). The FCS Express 7 Research Edition 7.18.0025 (De Novo Software, Los Angeles, CA, USA) was used for data analysis.
Expanded cells (4 × 105 in each group) were incubated in cold PBS containing 0.1% ChromPure Human IgG for 30 min. Then, the cells were incubated in the dark with primary antibodies at 4 °C for 30 min. The primary antibodies used are listed in Table 1. Fluorescence was analyzed by a BD FACSCalibur™ flow cytometer (BD Biosciences). Data analysis was performed by the FCS Express 7 Research Edition (De Novo software).
Total RNAs from the samples (n = 3), extracted using Trizol® (Millipore Sigma), were quantified, and about 2 μg of total RNA were used for reverse transcription with a high-capacity cDNA reverse transcription kit at 37 °C for 120 min, as recommended by the manufacturer (Thermo Fisher Scientific). A TaqMan® real-time quantitative polymerase chain reaction (qPCR) was used to evaluate the expression of stemness-related genes (MYC, KLF4, BMI1, POU5F1, NES, NOV, NANOG, and SOX2), adipogenic-related genes (LPL, FABP4, and CEBPA), osteogenic-related genes (BGLAP, ALPL, and COL1A1), and chondrogenic marker-related genes (SOX9, ACAN, Col2A1, PRG4, FBLN1, and FOXF1). GAPDH was utilized as the endogenous control gene. The TaqMan® Assay IDs of the primers are listed in Table 2. Each experiment was repeated three times using the Applied BiosystemsTM 7500 fast real-time PCR system (Applied Biosystems, Waltham, MA, USA). Relative transcript levels were calculated as χ = 2−ΔΔCt, in which ΔΔCt = ΔE − ΔC, ΔE = Ctexp − CtGAPDH, and ΔC = Ctct1 − CtGAPDH.

2.4. Immunofluorescence Staining of dECMs

The dECMs on pre-coated tissue culture coverslips were blocked with 1% bovine serum albumin (BSA) (Thermo Fisher Scientific) for 1 h and then incubated with primary antibodies (Table 3). Anti-type IV collagen (COL4), fibronectin (FN1), and nidogens 1 and 2 (NID1 and NID2) were detected using Alexa Fluor Plus 555-conjugated anti-mouse secondary antibodies (Invitrogen). Anti-laminin (LAMA) and anti-perlecan (PLC) were detected by Alexa Flour 488-conjugated anti-rabbit (Invitrogen) and Alexa Flour 488-conjugated anti-rat (Invitrogen) secondary antibodies, respectively. Fluorescent intensity was observed under a Zeiss Axiovert 40 CFL inverted microscope (Zeiss Oberkochen, Germany) using a 20× objective lens.

2.5. Induction and Assessment of Adipogenesis, Osteogenesis, and Chondrogenesis

For chondrogenic induction, 0.3 × 106 expanded cells from each group were centrifuged at 1200 rpm for 7 min in a 15-mL polypropylene tube to make a pellet. After 24 h of incubation in complete medium, D0 pellets were collected. The remaining pellets were cultured in a serum-free chondrogenic induction medium consisting of high-glucose Dulbecco’s Modified Eagle’s medium (DMEM), 40 μg/mL proline (Millipore Sigma), 100 μM dexamethasone (Millipore Sigma), 1× PSF, 0.1 mM ascorbic acid-2-phosphate (Wako Chemicals), and 1 × ITSTM Premix (BD Biosciences), with the supplementation of 10 ng/mL of transforming growth factor beta 3 (TGF-β3; PeproTech Inc., Rocky Hill, NJ, USA) in a 37 °C, 5% O2, 5% CO2 humidified incubator for 21 days. Chondrogenic differentiation was evaluated using histology and immunohistochemistry (IHC) analyses, as well as the qPCR targeting of chondrogenic-related genes (Table 2).
For histology, the representative pellets (n ꞊ 3) were fixed in 4% paraformaldehyde at 4 °C overnight, followed by dehydration in a gradient ethanol series, clearing with xylene, and embedding in paraffin blocks. For histological staining, 5-μm-thick sections were stained with Alcian blue (Thermo Fisher Scientific) for sulfated glycosaminoglycan (GAG) and counterstained with fast red for nuclei. For IHC analysis, the consecutive sections were probed with primary antibodies against type I collagen (GeneTex, Irvine, CA, USA), type II collagen (Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA, USA), and type X collagen (GeneTex), followed by the secondary antibody of biotinylated horse anti-mouse/rabbit IgG (H + L) (Vector, Burlingame, CA, USA). Immunoactivity was detected using Vectastain ABC reagent (Vector), with 3, 3′-diaminobenzidine (DAB, Vector) as a substrate. Counterstaining was performed with hematoxylin (Vector).
Expanded cells seeded in T25 flasks were incubated in an adipogenic induction cocktail, which comprised complete medium supplemented with 1 μM dexamethasone (Millipore Sigma), 0.5 mM isobutyl-1-methylxanthine (Thermo Fisher Scientific), 200 μM indomethacin (Millipore Sigma), and 10 μM insulin (BioVendor, Asheville, NC, USA) in a 37 °C incubator containing 5% CO2. For staining the lipid-filled droplets inside the cells after 21-day induction, the samples (n = 3) were fixed in 4% formaldehyde for 30 min and then stained with fresh 0.6% (w/v) Oil Red O (ORO) (Millipore Sigma) solution (60% isopropanol, 40% water) for 30 min before taking pictures using an Olympus IX51 microscope (Olympus America Inc., Center Valley, PA, USA). Adipogenic marker gene expression was quantified using qPCR (Table 2).
Expanded cells, grown in T25 flasks, were incubated in osteogenic medium, which was complete medium supplemented with 10 nM dexamethasone (Millipore Sigma), 50 mg/L L-ascorbic acid-2-phosphate (Wako Chemicals), and 10 mM β-glycerophosphate (glycerol 2-phosphate disodium salt hydrate) (Millipore Sigma) in a 37 °C incubator containing 5% CO2 for an additional 21 days. For the assessment of calcium deposition and matrix mineralization, induced cells (n = 3) were fixed with 4% formaldehyde for 30 min and then incubated in 1% Alizarin Red S (ARS) solution (pH = 4.3; Millipore Sigma) for 20 min before taking pictures using an Olympus IX51 microscope (Olympus America Inc.). Osteogenic marker gene expression was quantified using qPCR (Table 2).

2.6. Statistical Analysis

We conducted an ANOVA first; if the result was significant, we then compared the treatments pairwise. All experiments were performed in triplicate. The results from qPCR are presented as the mean ± SD. All statistical analyses were performed with the SPSS 20.0 statistical software (SPSS Inc., Chicago, IL, USA). p < 0.05 was considered to be statistically significant.

3. Results

3.1. Assessment of Stemness-Related Gene Expression in Fetal Stem Cells after Expansion on dECMs

After the preparation of dECMs (Figure 1A), the fetal MSCs seeded on either Plastic or dECMs for one day exhibited a polarized distribution (Figure 1B). qPCR analysis (Figure 1C) showed that Plastic-expanded fNPCs exhibited less expression of SOX2, NANOG, POU5F1, NES, and KLF4 than the corresponding fSDSCs, except for the expression of NOV, MYC, and BMI1. dECM expansion dramatically increased the mRNA levels of SOX2, NANOG, POU5F1, NOV, and NES in fNPCs, particularly for the first four genes in the SECM group and the last gene in the NECM group. Interestingly, dECM expansion diminished the expression of SOX2, NANOG, POU5F1, NOV, and KLF4 but increased the expression of NES and MYC in fSDSCs. Despite their being less of a response of fNPCs to dECM expansion in the expression of KLF4 and MYC, the fSDSCs exhibited an upregulation of MYC expression following dECM expansion, particularly for the NECM group.

3.2. Assessment of Surface Marker Expression in Fetal Stem Cells after Expansion on dECMs

To characterize surface marker expression and evaluate the cell proliferation capacity, flow cytometry was used to measure the surface phenotypes CD73, CD90, CD105, CD146, and SSEA4, as well as relative EdU incorporation in fetal stem cells following expansion on dECMs (Figure 2). We found that the percentage of CD73, CD90, CD146, and SSEA4 in fSDSCs remained stable in a range between 97% and 100% after growth on dECMs. In the fNPC groups, CD73 and CD90 remained stable at similar levels to the fSDSC groups. However, the percentage of CD146 was 95% in the Plastic group, which dropped to 65% in the NECM group and 82% in the SECM group. Interestingly, the percentage of SSEA4 was 50% in the PL group and increased to 65% in the NECM group and 78% in the SECM group. The above data indicate that fSDSCs exhibit a higher expression of MSC surface markers and a less marked response to dECM expansion compared to fNPCs. Intriguingly, the median fluorescent intensity (MFI) in both fetal stem cells showed the same trend in response to dECM expansion. Following expansion on dECMs, the MFI of CD73 and SSEA4 increased, while that of CD90 and CD146 decreased. Compared to the different responses of fNPCs and fSDSCs in the percentage of CD105 expression, dECM expansion increased relative EdU incorporation in both fetal cells, particularly for the SECM group.

3.3. Chondrogenic Capacity of Fetal Stem Cells after Growth on dECMs

To characterize the chondrogenic potential of fetal MSCs following expansion on dECMs, P5 fSDSCs and fNPCs grown on SECM and NECM were incubated in a pellet culture system to move toward chondrogenesis. After a 21-day chondrogenic induction, the qPCR data showed that fNPCs exhibited a greater response to chondrogenic induction than fSDSCs. fNPCs yielded higher expression levels of ACAN, COL2A1, and PRG4 but lower expression levels of COL1A1 and COL10A1 after expansion on NECM; fSDSCs yielded lower expression levels of ACAN and COL1A1 and higher expression levels of COL2A1 and COL10A1 after expansion on SECM (Figure 3A). Compared to Plastic expansion, interestingly, dECM-expanded fNPCs exhibited a decrease in FBLN1 (a marker for human articular cartilage) and an increase in FOXF1 (a marker for human NP tissue) [24], particularly with NECM; dECM-expanded fSDSCs displayed no significant change in FBLN1 and FOXF1 (Figure 3B).
Compared to Plastic expansion, dECM-expanded fNPCs yielded significantly larger pellets, while dECM-expanded fSDSCs did not show much change (Figure 4). dECM-expanded fNPCs yielded pellets with more intense staining of sulfated GAGs and type II collagen and less intense staining of type I collagen than the Plastic group; dECM-expanded fSDSCs yielded pellets with similar staining intensity to the Plastic group (Figure 4). Interestingly, there were no detectable differences in the intensity of type X collagen staining among all groups (Figure 4).
Applsci 14 01932 g003
Figure 3. Chondrogenic gene evaluation of fetal MSCs following expansion on dECMs. fNPCs (N) and fSDSCs (S), following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), were incubated in a pellet culture system with chondrogenic medium for 21 days. TaqMan® real-time PCR was used to evaluate the chondrogenic marker genes SOX9, ACAN, COL2A1, PRG4, COL1A1, and COL10A1 (A), and FBLN1 and the NPC marker gene FOXF1 (B) in the pellets collected at days 0, 10, and 21 after chondrogenic induction. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group. $ indicates a statistically significant difference from the corresponding PL group of day 0 pellets (p < 0.05).
Figure 3. Chondrogenic gene evaluation of fetal MSCs following expansion on dECMs. fNPCs (N) and fSDSCs (S), following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), were incubated in a pellet culture system with chondrogenic medium for 21 days. TaqMan® real-time PCR was used to evaluate the chondrogenic marker genes SOX9, ACAN, COL2A1, PRG4, COL1A1, and COL10A1 (A), and FBLN1 and the NPC marker gene FOXF1 (B) in the pellets collected at days 0, 10, and 21 after chondrogenic induction. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group. $ indicates a statistically significant difference from the corresponding PL group of day 0 pellets (p < 0.05).
Applsci 14 01932 g003
Applsci 14 01932 g004
Figure 4. Chondrogenic staining evaluation of fetal MSCs following expansion on dECMs. fNPCs (N) and fSDSCs (S), following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), were incubated in a pellet culture system with chondrogenic medium for 21 days. Alcian blue staining (Ab) was used to detect sulfated GAGs and immunohistochemistry staining (IHC) was used to detect type I collagen (COL1), type II collagen (COL2), and type X collagen (COLX). Scale bar: 400 μm.
Figure 4. Chondrogenic staining evaluation of fetal MSCs following expansion on dECMs. fNPCs (N) and fSDSCs (S), following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), were incubated in a pellet culture system with chondrogenic medium for 21 days. Alcian blue staining (Ab) was used to detect sulfated GAGs and immunohistochemistry staining (IHC) was used to detect type I collagen (COL1), type II collagen (COL2), and type X collagen (COLX). Scale bar: 400 μm.
Applsci 14 01932 g004

3.4. Adipogenic Capacity of Fetal Stem Cells after Growth on dECMs

To characterize the adipogenic potential of fetal MSCs following expansion on dECMs, P5 fSDSCs and fNPCs, grown on SECM and NECM for one passage, were incubated toward adipogenesis for 21 days. The qPCR data (Figure 5A) showed that following adipogenic induction, the fNPCs exhibited less expression/response than the corresponding fSDSCs. Compared to Plastic expansion, fSDSCs grown on SECM exhibited significantly greater levels of LPL, FABP4, and CEBPA, whereas fSDSCs grown on NECM only displayed a higher level of FABP4. The fNPC groups displayed no significant increases in any adipogenic marker gene expression. These trends are supported by an assessment of adipogenic markers at the protein level, such as that of PLIN2 and FABP4 by Western blot analysis (Figure 5B) and of lipid droplets by ORO staining (Figure 5C).

3.5. Osteogenic Capacity of Fetal Stem Cells after Growth on dECMs

To characterize the osteogenic potential of fetal MSCs following expansion on dECMs, P5 fSDSCs and fNPCs, grown on SECM and NECM for one passage, were incubated toward osteogenesis for 21 days. The qPCR data (Figure 6A) showed that after osteogenic induction, the fNPCs responded more significantly than the corresponding fSDSCs and exhibited a higher level of expression of BGLAP and ALPL. Compared to the Plastic group, dECM-expanded fNPCs exhibited less BGLAP expression, whereas the dECM-expanded fSDSCs displayed higher ALPL expression. These mRNA data were in line with the protein expression of ALPL, determined by Western blot analysis (Figure 6B), and calcium deposition, determined by Alizarin Red S staining (Figure 6C).

3.6. Major Matrix Protein Expression in Fetal Stem Cells, Their Matrix, and Three-Lineage Differentiated Cells

Immunofluorescence staining analysis showed that the NECM and SECM were positively stained for FN1, PLC, NID1, NID2, LAMA, and COL4. The dECMs from both cells exhibited a comparable density of staining in NID1, NID2, and COL4, except that PLC was intensely stained in SECM and FN1 and LAMA were intensely stained in NECM (Figure 7A). The intensity of dECM staining was in line with the qPCR data of expanded fetal stem cells, in which Plastic-expanded fNPCs expressed more FN1 and LAMA5 and Plastic-expanded fSDSCs expressed more HSPG2 (Figure 7B). We found that after chondrogenic induction, FN1 and HSPG2 were upregulated and NID1, NID2, LAMA5, and LAMB1 were downregulated in the fNPCs, while FN1 was upregulated and HSPG2, NID1, NID2, LAMA5, and LAMB1 were downregulated in the fSDSCs (Figure 7B). After adipogenic induction, FN1, HSPG2, and LAMA5 were downregulated in the fNPCs, while NID2 was upregulated and FN1, HSPG2, NID1, LAMA5, and LAMB1 were downregulated in the fSDSCs (Figure 7B). After osteogenic induction, FN1, HSPG2, NID1, NID2, LAMA5, and LAMB1 were upregulated in the fNPCs, while FN1, HSPG2, NID1, NID2, and LAMB1 were upregulated in the fSDSCs (Figure 7B).

4. Discussion

Fetal MSCs are a promising approach to stem-cell therapy in regenerative orthopedic medicine [25]. An ex vivo environment, such as hypoxia, has been used to modulate the regenerative potential of fetal stem cells [23]. Knowing that a matrix microenvironment can influence the therapeutic potential of adult stem cells [11,26], in this study, we characterized two fetal cell types for their response to expansion on fetal dECM and the subsequent differentiation capacity.
Compared to fNPCs, the fSDSCs showed a higher overall expression of MSC surface markers, indicating greater cell proliferation capacity. Interestingly, fSDSCs grown on SECM exhibited an increased expression of CD73, SSEA4, and relative EdU incorporation but a decreased expression of CD90, CD105, and CD146. This finding is in line with previous reports, in which aSDSCs or fSDSCs grown on SECMs and deposited by either aSDSCs or fSDSCs displayed a decrease in expression of CD90 and CD105 but an increase in SSEA4 [19,27]. An increase in the expression of SSEA4 and relative EdU incorporation indicates that dECM expansion promoted MSC proliferation. The International Society for Cellular Therapy’s 2006 conference defined CD73, CD90, and CD105 as surface markers for human MSCs [28]. Given this definition, it is challenging to interpret the similar trend in the expression of MSC surface markers following dECM expansion with the dissimilar differentiation capacities shown in this study and others [29]. We also found that fetal cells responded differently to matrix rejuvenation in stemness gene expression. While fSDSCs are considered closer to MSCs and, therefore, more pluripotent than fNPCs, after dECM rejuvenation, interestingly, the fNPCs displayed higher levels of pluripotency gene expression than the fSDSCs.
Following expansion on Plastic flasks, different from aSDSCs losing their chondrogenic capacity during passaging from P3 to P5 [30], the fSDSCs regained their chondrogenic potential from P2 to P9 [27]. dECM rejuvenation is of benefit to adult MSCs grown on fetal dECM [19] and fetal MSCs grown on adult dECM [27] but not to adult MSCs expanded on old dECM [20] and fetal MSCs expanded on fetal dECM [27]. In this study, fSDSCs grown on dECMs deposited by fSDSCs and fNPCs yielded 21-day pellets with a decreased expression of SOX9 and ACAN and increased expression of COL2A1, with no significant difference in PRG4 and FBLN1. In contrast, fNPCs grown on dECMs and deposited by fSDSCs and fNPCs yielded 21-day pellets with an increased expression of ACAN, COL2A1, and PRG4, and a decreased expression of COL1A1 and COL10A1. This finding indicates that, despite the fact that both are fetal cells, fNPCs (a progenitor cell for NP tissue) responded more positively to the rejuvenation of dECMs deposited by fSDSCs and fNPCs than fSDSCs. Interestingly, dECM-rejuvenated fNPCs exhibited a less significant response to both osteogenic and adipogenic induction compared to the Plastic group, which may be associated with the inherent nature of NPCs [31]. Compared to fNPCs, fSDSCs are closer to fetal MSCs. Despite less of a response to chondrogenic and osteogenic induction following dECM rejuvenation, under adipogenic induction, fSDSCs grown on dECM deposited by fSDSCs exhibited enhanced adipogenic capacity, as evidenced by the greater expression of LPL, FABP4, and CEBPA by qPCR analysis and of PLIN2 and FABP4 by Western blot analysis, as well as the greater intensity of ORO staining indicating lipid droplets, which is in line with a previous report [27].
Major matrix components in dECMs and dynamic expression during three-lineage induction might contribute to lineage differentiation [32,33,34,35]. For instance, the intense staining of fibronectin in NECM might contribute to greater levels of chondrogenic marker expression in the fNPC group, compared to the fSDSC group [34,36]. Since fibronectin inhibits adipogenic differentiation [37,38,39], a lower level of fibronectin expression might contribute to higher levels of adipogenic differentiation in the fSDSC group. Enhanced chondrogenic capacity in dECM-expanded fNPCs might also contribute by the upregulation of LAMA5, which has been shown to favor chondrogenesis [33]. Interestingly, fSDSCs did not show a significant change in LAMA5 expression during osteogenic induction, indicating that LAMA5 might contribute to the osteogenic differentiation of fNPCs.
In summary, we demonstrated that matrix rejuvenation influences fetal stem cell stemness, proliferation, and differentiation capacities, establishing the potential of the modulating matrix environment to enhance fetal stem cell efficacy as a source for orthopedic regenerative stem cell therapy and tissue engineering.

Author Contributions

Conceptualization, M.P.; methodology, Y.A.P.; validation, Y.A.P.; formal analysis, Y.A.P.; investigation, Y.A.P., J.P. and M.P.; resources, Y.A.P.; data curation, Y.A.P.; writing—original draft preparation, Y.A.P., J.P. and M.P.; writing—review and editing, Y.A.P. and M.P.; software, Y.A.P.; supervision, M.P.; project administration, M.P.; funding acquisition, M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially supported by grants from the National Institutes of Health, United States (Grant No. AR078846 and AR067747).

Institutional Review Board Statement

Ethical review and approval were waived for this study, due to the commercial purchase of stem cells from unidentified donors.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We thank Suzanne Danley for editing the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Fetal MSC morphology and stemness gene expression following expansion on dECMs. (A) Before and after the decellularization of ECMs deposited by fNPCs and fSDSCs. Scale bar: 100 μm. (B) Cell morphology of fetal MSCs after expansion on dECMs deposited by fNPCs (NE) or fSDSCs (SE), with tissue culture plastic (PL) as a control. Scale bar: 100 μm. (C) Assessment of stemness gene expression, including SOX2, NANOG, POU5F1, NOV, NES, KLF4, MYC, and BMI1 in fNPCs and fSDSCs, following expansion on NE, SE, or PL using TaqMan® real-time qPCR. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding NE group. $ indicates a statistically significant difference from the corresponding PL group of fNPCs (p < 0.05).
Figure 1. Fetal MSC morphology and stemness gene expression following expansion on dECMs. (A) Before and after the decellularization of ECMs deposited by fNPCs and fSDSCs. Scale bar: 100 μm. (B) Cell morphology of fetal MSCs after expansion on dECMs deposited by fNPCs (NE) or fSDSCs (SE), with tissue culture plastic (PL) as a control. Scale bar: 100 μm. (C) Assessment of stemness gene expression, including SOX2, NANOG, POU5F1, NOV, NES, KLF4, MYC, and BMI1 in fNPCs and fSDSCs, following expansion on NE, SE, or PL using TaqMan® real-time qPCR. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding NE group. $ indicates a statistically significant difference from the corresponding PL group of fNPCs (p < 0.05).
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Figure 2. Assessment of fetal MSC surface marker expression following expansion on dECMs. Flow cytometry was used to measure the expression of the MSC surface markers CD73, CD90, CD105, CD146, and SSEA4, as well as relative EdU incorporation into fNPCs and fSDSCs following expansion on dECM deposited by fetal NPCs (NE) or fetal SDSCs (SE), with tissue culture plastic (PL) as a control.
Figure 2. Assessment of fetal MSC surface marker expression following expansion on dECMs. Flow cytometry was used to measure the expression of the MSC surface markers CD73, CD90, CD105, CD146, and SSEA4, as well as relative EdU incorporation into fNPCs and fSDSCs following expansion on dECM deposited by fetal NPCs (NE) or fetal SDSCs (SE), with tissue culture plastic (PL) as a control.
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Figure 5. Adipogenic evaluation of dECM-expanded fetal MSCs. Following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (S), or tissue culture plastic (PL), the fNPCs (N) and fSDSCs (S) were incubated in an adipogenic medium for 21 days. (A) TaqMan® real-time qPCR was used to evaluate the adipogenic marker genes LPL, FABP4, and CEBPA in day 21 samples. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group. $ indicates a statistically significant difference from the corresponding PL group of cell samples (p < 0.05). (B) Western blot analysis was used to evaluate the adipogenic markers PLIN2 and FABP4 of day 21 samples. GAPDH serves as the internal control. (C) Oil Red O staining was used to detect the lipid droplets of adipogenically induced cells in T25 flasks. Scale bar: 100 μm.
Figure 5. Adipogenic evaluation of dECM-expanded fetal MSCs. Following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (S), or tissue culture plastic (PL), the fNPCs (N) and fSDSCs (S) were incubated in an adipogenic medium for 21 days. (A) TaqMan® real-time qPCR was used to evaluate the adipogenic marker genes LPL, FABP4, and CEBPA in day 21 samples. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group. $ indicates a statistically significant difference from the corresponding PL group of cell samples (p < 0.05). (B) Western blot analysis was used to evaluate the adipogenic markers PLIN2 and FABP4 of day 21 samples. GAPDH serves as the internal control. (C) Oil Red O staining was used to detect the lipid droplets of adipogenically induced cells in T25 flasks. Scale bar: 100 μm.
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Figure 6. Osteogenic evaluation of dECM-expanded fetal MSCs. Following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), the fNPCs (N) and fSDSCs (S) were incubated in an osteogenic medium for 21 days. (A) TaqMan® real-time qPCR was used to evaluate the osteogenic marker genes BGLAP and ALPL of day 21 samples. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group. $ indicates a statistically significant difference from the corresponding PL group of cell samples (p < 0.05). (B) Western blot analysis was used to evaluate the osteogenic marker ALPL of day 21 samples. GAPDH serves as the internal control. (C) Alizarin Red S staining was used to detect the calcium deposition of osteogenically induced cells in T25 flasks. Scale bar: 100 μm.
Figure 6. Osteogenic evaluation of dECM-expanded fetal MSCs. Following expansion “on” dECMs deposited by fNPCs (NE), fSDSCs (SE), or tissue culture plastic (PL), the fNPCs (N) and fSDSCs (S) were incubated in an osteogenic medium for 21 days. (A) TaqMan® real-time qPCR was used to evaluate the osteogenic marker genes BGLAP and ALPL of day 21 samples. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group. $ indicates a statistically significant difference from the corresponding PL group of cell samples (p < 0.05). (B) Western blot analysis was used to evaluate the osteogenic marker ALPL of day 21 samples. GAPDH serves as the internal control. (C) Alizarin Red S staining was used to detect the calcium deposition of osteogenically induced cells in T25 flasks. Scale bar: 100 μm.
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Figure 7. Major matrix protein expression in dECMs and differentiated fetal MSCs. (A) Immunofluorescence staining was used to detect the expression of FN1, PLC, NID1, NID2, LAMA, and COL4 in dECMs deposited by fNPCs or fSDSCs. Scale bar: 100 μm. (B) TaqMan® real-time qPCR was used to evaluate the mRNA levels of FN1, HSPG2, NID1, NID2, LAMA5, and LAMB1 in expanded fetal MSCs and in chondrogenically, adipogenically, and osteogenically induced cells. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group (p < 0.05). $ indicates a statistically significant difference from the corresponding PL expanded cell samples (p < 0.05).
Figure 7. Major matrix protein expression in dECMs and differentiated fetal MSCs. (A) Immunofluorescence staining was used to detect the expression of FN1, PLC, NID1, NID2, LAMA, and COL4 in dECMs deposited by fNPCs or fSDSCs. Scale bar: 100 μm. (B) TaqMan® real-time qPCR was used to evaluate the mRNA levels of FN1, HSPG2, NID1, NID2, LAMA5, and LAMB1 in expanded fetal MSCs and in chondrogenically, adipogenically, and osteogenically induced cells. GAPDH served as the internal control. Data are shown as a bar chart. * indicates a statistically significant difference from the corresponding PL group (p < 0.05). # indicates a statistically significant difference from the corresponding fNPC group (p < 0.05). $ indicates a statistically significant difference from the corresponding PL expanded cell samples (p < 0.05).
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Table 1. Primary antibodies used in the surface marker analysis.
Table 1. Primary antibodies used in the surface marker analysis.
AntibodyCompany InformationConcentrationCatalog No.
CD73 Monoclonal Antibody (AD2), APC, humaneBioscienceTM, Fisher Scientific, Waltham, MA, USA0.125 μg/test17-0739-42
Anti-CD146 Monoclonal Antibody (P1H12) PE, humaneBioscienceTM, Fisher Scientific0.125 μg/test12-1469-42
PE anti-human SSEA-4BioLegend, Dedham, MA, USA0.125 μg/test330406
CD90-APC-Vio® 770, humanMiltenyi Biotec, San Diego, CA, USA2 μL/test130-114-863
CD105-PerCp-Vio® 700, humaneBioscienceTM2 μL/test130-112-170
Table 2. TaqMan® assay ID information of the target genes for qPCR.
Table 2. TaqMan® assay ID information of the target genes for qPCR.
Gene NameFull NameTaqMan® Assay ID
Stemness-related genes
MYCMYC proto-oncogeneHs00153408_m1
KLF4Kruppel-like factor 4Hs00358836_m1
BMI1B lymphoma Mo-MLV insertion region 1 homologHs00180411_m1
POU5F1POU class 5 homeobox 1Hs04260367_gH
NESNestinHs04187831_g1
NOVNephroblastoma overexpressedHs00159631_m1
NANOGNanog homeoboxHs02387400_g1
SOX2SRY-box2Hs01053049_s1
Adipogenesis-related genes
LPLLipoprotein lipaseHs00173425_m1
FABP4Fatty acid-binding protein 4Hs01086177_m1
CEBPACCAAT/enhancer-binding protein alphaHs00269972_s1
Osteogenesis-related genes
BGLAPBone gamma-carboxyglutamate proteinHs01587814_g1
ALPLAlkaline phosphatase, liverHs01029144_m1
COL1A1Type I collagenHs00164004_m1
Chondrogenesis-related genes
SOX9SRY-Box 9Hs00165814_m1
ACANAggrecanHs00153936_m1
Col2A1Type II collagenHs00156568_m1
PRG4Proteoglycan 4Hs00981633_m1
FBLN1Fibulin 1Hs00972609_m1
FOXF1Forkhead box F1Hs00230962_m1
Housekeeping internal gene
GAPDHGlyceraldehyde-3-phosphate dehydrogenaseHs02758991_g1
Table 3. Primary antibodies used in the immunofluorescence staining of dECMs.
Table 3. Primary antibodies used in the immunofluorescence staining of dECMs.
AntibodyCatalog No.CompanySpeciesWorking Conc.
Type II collagenII6B3-cDevelopmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USAMouse3 μg/mL
Type IV collagenM3F7Mouse3 μg/mL
FibronectinHFN 7.1Mouse3 μg/mL
LamininPA1-16730Invitrogen, Waltham, MA, USARabbit20 μg/mL
Col1A1 (3G3)sc-293182Santa Cruz Biotechnology, Inc., Dallas, TX, USAMouse1 μg/mL
Perlecan (A7L6)sc-33707Rat4 μg/mL
Nidogen 1 (C-7)sc-133175Mouse2 μg/mL
Nidogen-2 (F-2)sc-377424Mouse2 μg/mL
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Pei, Y.A.; Patel, J.; Pei, M. Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells. Appl. Sci. 2024, 14, 1932. https://doi.org/10.3390/app14051932

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Pei YA, Patel J, Pei M. Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells. Applied Sciences. 2024; 14(5):1932. https://doi.org/10.3390/app14051932

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Pei, Yixuan Amy, Jhanvee Patel, and Ming Pei. 2024. "Extracellular Matrix Tunes the Regenerative Potential of Fetal Stem Cells" Applied Sciences 14, no. 5: 1932. https://doi.org/10.3390/app14051932

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