High‐efficient generation of VCAM‐1+ mesenchymal stem cells with multidimensional superiorities in signatures and efficacy on aplastic anaemia mice

Abstract Objective Longitudinal studies have indicated VCAM‐1+ mesenchymal stem/stromal cells (MSCs) as promising resources in regenerative medicine, yet the abundance in gene expression is far from adequate in the advantaged and “discarded” hUC‐MSCs. Thus, high‐efficient preparation and systematic dissection of the signatures and biofunctions of the subpopulation is the prerequisite for large‐scale clinical applications. Materials and methods We primarily took advantage of a cytokine‐based programming strategy for large‐scale VCAM‐1+ hUC‐MSC generation (III‐MSCs). Thereafter, we conducted multifaceted analyses including cytomorphology, immunophenotype, cell vitality, multilineage differentiation, whole‐genome analysis, tube formation and Matrigel plug assay, lymphocyte activation and differentiation, and systemic transplantation for aplastic anaemia (AA) treatment. Results III‐MSCs with high‐proportioned VCAM‐1 expression were obtained by combining IL‐1β, IL‐4 with IFN‐γ, which exhibited comparable immunophenotype with untreated hUC‐MSCs (NT‐MSCs) but revealed multidimensional superiorities both at the cellular and molecular levels. Simultaneously, systemic infusion of III‐MSCs could significantly ameliorate clinicopathological features and finally help facilitate haematopoietic reconstruction and immunoregulation in AA mice. Conclusions We have established a high‐efficient procedure for large‐scale generation of III‐MSCs with preferable signatures and efficacy upon aplastic anaemia in mice. Our findings suggested that III‐MSCs were advantageous sources with multifaceted characteristics for regenerative medicine.


| INTRODUC TI ON
Mesenchymal stem/stromal cells (MSCs) are acknowledged as a heterogeneous population and the most important stromal cells in the niche for haematopoiesis and coordinate contribution to regenerative medicine. [1][2][3] For decades, we and other investigators have reported the establishment of MSCs from a various range of adult tissues such as bone marrow, adipose tissue, dental pulp and even human pluripotent stem cells (hPSCs). 2,[4][5][6] Differ from the adult tissue-derived counterparts, MSCs extracted from perinatal tissues (eg, umbilical cord, placenta) have been proved with preferable characteristics in multifaceted signatures such as juvenility and easy preparation without acquired pollution or ethical risk, and in particular, the hUC-MSCs with vigorously long-term reproductive capacity and better cellular pharmaceutical prospects. 2 Simultaneously, there is a suspicious attitude towards the therapeutic effect and variability in quality in considering the allogeneic cell sources and the probability of genetic variability. 7,8 For instance, we and Zhang, et al recently reported the multidimensional alterations in efficacy on acute graft-versus-host disease and acute liver failure, respectively. 2,9 Moreover, it is worth noting that functional heterogeneity for MSCbased cytotherapy has attracted the attention of investigators both in the field of fundamental and translational research, which indicates the urgent need to distinguish alternatively inexhaustible subpopulations of MSCs. 10,11 To date, limited surface biomarkers have been developed for dissecting the subpopulations with potentially unique bioactivity. Distinguish from the conventionally heterogeneous MSCs, a novel MSCA-1 + CD56 + subset exhibited predominant differentiation potential towards chondrocytes and pancreatic-like islets. 12 Consistently, the enriched CD56 + subset from multiclonal BM-MSCs resulted in enhanced chondrogenesis and bone regeneration. 13 In recent years, we have originally demonstrated the possibility of generating a highly bioactive subpopulation from the heterogeneous MSCs, especially for high-quality VCAM-1 + (CD106 + ) hUC-MSCs. 14,15 In general, compared with the CD106subset, increased proangiogenic potential, preferable immunomodulatory property and enhanced homing capacity of CD106 + MSCs were observed, which collectively indicated the promising applications in cellular therapeutics. 14,16 However, the expression abundance of VCAM-1 is far from adequate in the advantaged and "discarded" hUC-MSCs.
Meanwhile, the deficiency of systematic and rigorous clarification on the biological and genetic signatures together with efficacy in vivo still hinders their prospects in regenerative medicine. Hence, the preparation and systematic dissection of the signatures and biofunctions of VCAM-1 + subpopulation is the prerequisite for large-scale clinical applications.
In this study, we have originally established a rapid and high-efficiency strategy for generating VCAM-1 + III-MSCs with multidimensional preponderances in signatures, together with highly conserved genome and superior efficacy for AA treatment. Taken together, we concluded that it was feasible to generate large-scale and homologous III-MSCs with preferable biological and genetic attributes together with therapeutic effects on aplastic anaemia from the "discarded" UC-MSCs for regenerative medicine.

| Cell culture and preconditioning
hUC-MSCs were cultured for further analyses as we previously described. 17,18 For III-MSC induction, the hUC-MSCs were preconditioned with the indicated cytokines and detailed procedures as listed in Table S1 and Supplementary Procedures.
The primer sequences of the indicated genes and detailed procedures were listed in Table S3 and Supplementary Procedures.

| Matrigel plug assay in vivo
The in vivo Matrigel plug assay was performed as we previously described. 15

| Aplastic anaemia (AA) model and MSC transplantation
The AA model was conducted as described with several modifications. 22 In brief, CByB6F1 recipients were pre-irradiated with 5 Gy

| High-efficient generation of III-MSCs with preferable multilineage differentiation capacity
Recently, we have originally demonstrated VCAM-1 as a novel biomarker of MSC subpopulation with unique attributes including superior proangiogenic and immunomodulatory properties. 14,15 However, the proportion of the VCAM-1 + subset is desperately inadequate for large-scale application in regenerative medicine. 26 Simultaneously, the rigorous and systematic evaluation of multidimensional signatures both at the cellular and molecular levels is dauntingly unprocurable.
For the purpose, we took advantage of a programming strategy to facilitate VCAM-1 + hUC-MSC generation by utilizing small-scale cytokine screening as we recently reported with modification. 5,19 Of them, we found IL-1β, IL-4 and IFN-γ could improve the generation of VCAM-1 + cells, respectively ( Figure 1A; Figure S1A-B). Thereafter, we further verified that the invariably maximum efficiency for inducing VCAM-1 + hUC-MSCs was 83.00%  Figure S1F).

F I G U R E 2
The distribution of differentially expressed genes and mutation spectrum in NT-mesenchymal stem/stromal cells (MSCs) and III-MSCs. A, Gene expression distribution of total genes in the genome of NT-and III-MSCs. B, Volcano plot analysis of total genes. C and D, GO analysis of upregulated (C) and downregulated (D) genes (P < .05, log 2 FC > 1). E, GSEA shows enrichment plot of the indicated subsets in NT-and III-MSCs. F and G, Distribution of SNPs (F) and fusion genes (G) in the genome of NT-and III-MSCs Additionally, to illuminate the potential influence of the indicated cytokine addition on the multilineage differentiation capacity, we took advantage of the procedure to differentiate NT-MSCs and III-MSCs towards adipocytes, osteoblasts and chondrocytes, respectively.

| III-MSCs showed conserved landscape of gene expression profiling together with certain alterations
To systematically dissect the potential alterations at molecular level, we turned to gene expression profiling of III-MSCs and NT-MSCs.
Generally, from the violin illustration and volcano plots, we found  (Figure 2A, B). Furthermore, we noticed the significantly upregulated genes were associated with multiple biological processes including metabolism and extracellular matrix, together with signal pathways (eg, PTK2, Ephrin) ( Figure 2C).

| III-MSCs manifested multitudinous similarities with NT-MSCs in cell vitality
Cell vitality and homing capacity are recognized as the fundamental for efficacy in regenerative medicine. 2,9 As shown by the heatmap diagram, a certain amount of cell proliferation-associated genes was upregulated in III-MSCs whereas no distinct tendency in cell cycleand apoptosis-associated genes ( Figure 3A; Table S4). In consist with the gene clustering spectrum, III-MSCs showed preferable prolifera-

| III-MSCs possessed preferable characteristics in migration and angiogenesis over NT-MSCs
Therewith, we were curious about whether migration and proangiogenic activities were influenced as well. GSEA analysis intuitively indicated that ECM-receptor interaction-, crosslinking collagen fibrils-and laminin interactions-associated gene sets were notably disparate, which were confirmed by assessments on cell migration by wound healing analysis ( Figure 4A-C).
Proangiogenic activity is an advantaged property of VCAM-1 + subpopulation in hUC-MSCs and hCV-MSCs. 14,15 Thus, we conducted the tubular network formation assay in vitro and found III- Collectively, these data revealed that III-MSCs had preponderances in migration and proangiogenesis both in vitro and in vivo. F I G U R E 3 III-mesenchymal stem/stromal cells (MSCs) with comparable cell vitality but enhanced CFU-F formation potential. A, Heatmap analysis of gene sets in NT-and III-MSCs. B, Pd assay of NT-and III-MSCs for 17 passages. C, Proliferation assay with CCK-8 kit. D and E, Distributions of apoptotic population in NT-and III-MSCs as shown by FCM diagram (D) and statistical analysis (E). F and G, The proportion of apoptotic population as shown by FCM diagram (F) and statistical analysis (G). H and I, Images of total CFU-Fs (H) and representative colonies (I) (scale bar = 500 μm). J, Statistical analysis of CFU-F numbers. Data were shown as mean ± SEM (n = 3). *P < .05, **P < .01; NS, not significant | 9 of 14 WEI Et al.

| III-MSCs exhibited superiorities in immunosuppressing proinflammatory cytokine secretion and lymphopoiesis in vitro
To explore the immunosuppressive signature of III-MSCs, we primarily analysed the expression pattern of inflammatory-associated cy-  Table S4). When compared with NT-MSCs, the inhibitory effect of III-MSCs on CD4 + T lymphocyte activation and differentiation towards Th1 and Th17 cells were enhanced by III-MSCs, whereas the abundance of Treg subpopulation was increased ( Figure 5D-K). In summary, our data indicated that III-MSCs exhibited reinforced immunoregulatory properties.

| III-MSCs displayed enhanced ameliorating effect on aplastic anaemia mice compare to NT-MSCs
Having illuminated the potential superiorities in vivo, we turned to factors, yet the production is far from enough. 29 Herein, we have established a programming strategy without gene editing for convenient III-MSC generation and systematical evaluation, which is the prerequisite for large-scale clinical applications.
State-of-the-art updates towards MSCs prompt the prospects and feasibility for application in disease treatment and health management, especially for recurrent and refractory disorders. 2,30,31 However, several studies argued that the efficacy of MSCs on rheumatology and intervertebral disc degeneration treatment or heart regeneration was suspicious and even controversial. 7,8,32 On the basis of analysing the root of the above-mentioned issue, the cell vitality and homing are acknowledged as the foremost elements need to be taken into consideration. 2,33,34 On the one hand, MSCs with diverse origins and culture conditions possess disparate signatures and biofunctions. For instance, hUC-MSCs and hPSC-MSCs have been demonstrated with preferable property in long-term in vitro proliferation, together with more robust immunoregulatory capacity. 2,5,19 However, MSCs at various passages or from different individuals could display alteration in multifaceted signatures and hereditary stability. 2,9,35 In this study, we also observed multifaceted variations in the efficacy on AA treatment between III-MSCs and NT-MSCs. On the other hand, increasingly researches attempt to verify the heterogeneity of MSCs by dissecting the concealed subpopulations, which is also the principal issue for standardizing and guiding therapeutic uses of MSCs. 11,36 In 2013, we successfully identified the VCAM-1 + subpopulation in MSCs with various origins and confirmed the splendid characteristics in proangiogenesis and immunoregulation by comparing CD106 + subpopulation with CD106cells. 14,15 However, the abundance of VCAM-1 is far from adequate in the advantaged and "discarded" hUC-MSCs without ethical risk and acquired influence. 14 Meanwhile, the proportion of VCAM-1 + MSCs in patients with bone marrow failure of acquired AA was sharply declined, which collectively confirmed the negative correlation and potential application with AA as well. 14 Taken together, our data provided a profound evidence for VCAM-1 + MSCs as a novel and pivotal subpopulation with multidimensional superiorities in signatures and efficacy, together with supplying overwhelmingly new references for explaining the above-mentioned existing dispute on MSC-based clinical therapeutics. Above all, even though VCAM-1 + hUC-MSCs have been high-efficiently manufactured by cytokine-based programming, yet there are extensive efforts associated with safety, effectiveness and repeatability need to be done before large-scale applications in cytotherapy.

CO N FLI C T O F I NTE R E S T
The authors declare there is no competing interest and all authors consent to publish the data.

AUTH O R CO NTR I B UTI O N S
YW, and LZ: collection and assembly of data, manuscript writing; YC, XR, YG, BS, CL, ZH: collection and assembly of data; YW, XR: MSC transplantation and collection of data; LZ, LZ, and ZH: conception and design, data analysis and interpretation, final approval of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article and its supplementary information files.
Meanwhile, the data sets used and analysed during the current study are also available from the corresponding author on reasonable request.