Co-Transplantation of Hypoxia Pretreated Human Adipose Derived Mesenchymal Stem Cells and Cord Blood Mononuclear Cells to Treat Rats With Acute Myocardial Infarction


 BackgroundHuman adipose derived mesenchymal stem cells (ASCs) are ideal candidates for the treatment of acute myocardial infarction (AMI), due to their favorable availability and regenerative potential. However, in vivo studies showed that ASCs are not resilient at the infarcted area, for a shortage of blood and oxygen supply. Hypoxic pretreatment was proven to be an effective way to enhance cell survival in ischemic atmosphere. Moreover, co-transplantation of stem cells was another promising strategy to improve cardiac function after transplantation. So, we hypothesized that hypoxic pretreated ASCs combined with proangiogenic cord blood mononuclear cells (CBMNCs) would promote treatment efficacy after co-transplantation.MethodsASCs extracted from male volunteer were preconditioned in hypoxic condition (HP-ASC) for 24h, and total RNA were extracted after that. Gene expressions were compared between HP-ASC and ASC. Then, we transplanted stem cells to female Wistar rats which divided into different groups: (1) HP-ASCs group (n=10, 1x106ASCs); (2) HP-ASCs + CBMNCs group (n=10, 0.5×106 ASCs+0.5×106 CBMNCs); (3) CBMNCs group (n=10, 1×106 ASCs); (4) Control group (n=10, 40μL PBS); (5) Sham group (n=10). Echocardiogram was performed before (0d) and after (30d) after cell transplantation. Hearts were harvested at 30d to analyze the infarct size, myocardium apoptosis, stem cells viability and angiogenesis. ResultsIn vitro study showed that HP-ASCs had a wide range of paracrine function, with the incretion growth factors and their receptors, which would support the cell survivals. In addition, HP-ASCs also gained potentials in hypoxic adaptation (increased expression of HO-1 and SDF-1), as well as homing and immigrating abilities (CXCR4, ICAM-1 and ICAM-2). In vivo studies showed that, 30 days after transplantation in AMI rats, the HP-ASCs group had a better improvement in cardiac function; reduction of the infarct size; and decrease of ASCs death than the other groups (HP-ASCs > HP-ASCs + CBMNCs ≧ CBMNCs > PBS) (p<0.05). However, the combined group of HP-ASCs and CBMNCs had more significant angiogenesis than the other groups (HP-ASCs + CBMNCs > CBMNCs > HP-ASCs > PBS) (p <0.05).ConclusionsHP-ASCs alone had a greater potential in improving cardiac function in AMI rats. However, the combination of HP-ASCs and CBMNCs had a better result in angiogenesis.


Abstract Background
Human adipose derived mesenchymal stem cells (ASCs) are ideal candidates for the treatment of acute myocardial infarction (AMI), due to their favorable availability and regenerative potential. However, in vivo studies showed that ASCs are not resilient at the infarcted area, for a shortage of blood and oxygen supply.

Material and methods
To solve the problem of living in the hypoxic environment, we accommodated ASCs within the hypoxic condition. To enhance the capillary system, we combined the hypoxic pretreated ASCs (HP-ASCs) with cord blood mononuclear cells (CBMNCs), which have a great potential for neovascularization. We hypothesized that this combination system would improve the transplantation e ciency.

Results
In vitro study showed that HP-ASCs had a wide range of paracrine function, with the incretion growth factors and their receptors, which would support the cell survivals. In addition, HP-ASCs also gained potentials in hypoxic adaptation (increased expression of HO-1 and SDF-1), as well as homing and immigrating abilities (CXCR4, ICAM-1 and ICAM-2). In vivo studies showed that, 30 days after transplantation in AMI rats, the HP-ASCs group had a better improvement in cardiac function; reduction of the infarct size; and decrease of ASCs death than the other groups (HP-ASCs > HP-ASCs + CBMNCs ≧ CBMNCs > PBS) (p<0.05). However, the combined group of HP-ASCs and CBMNCs had more signi cant angiogenesis than the other groups (HP-ASCs + CBMNCs > CBMNCs > HP-ASCs > PBS) (p <0.05).
Conclusions HP-ASCs alone had a greater potential in improving cardiac function in AMI rats. However, the combination of HP-ASCs and CBMNCs had a better result in angiogenesis.

Background
Myocardial infarction (MI) is a life-threatening emergency which causes hospitalization and life span reduction in US and worldwide [1]. The standard treatment of MI, including catheterization and medication, has shown signi cant improvement in outcomes. However, the damaged heart tissues can only be repaired with scars, which lost the myocardium characters of conductivity and contractility.
Consequently, the brotic scar leads to a chronic process of cardiac remodeling, which results in compromised ventricular performance and chronic heart failure. The positive correlation between the size of infarction and mortality urged us to nd a new treatment approach for heart regeneration [2].
Physiologically, the hearts of new-born babies have partial regenerative potential through the proliferation of pre-existing cardiomyocytes (CMs), but this does not exist in adults [3]. Recently, numerous studies reported that stem/ progenitor cells can be used for the treatment of ischemic heart diseases [4,5].
Among those, adipose-derived stromal cells (ASCs) have shown encouraging outcomes in clinical trials [6,7]. ASCs have more priorities than bone marrow stem cells for clinical use [8,9], for their large quantities in isolation and minimal invasion, as well as high yield of progenitor cells per volume.
However, there are still some obstacles in improving ASCs survivals after transplantation, which result from regional hostile microenvironment. The eld is not suitable for transplanted cells to survive, as it is surrounded by necrotic cells and in ammatory cells that increase the oxidative stress. As a consequence, stem cells generally display limited survival and low retention rate in injured tissues, reducing the bene t of their therapeutic effects [10]. Therefore, we need to solve the problem of hypoxia adaption and blood supply, in order to elongate the cell lives in vivo.
To overcome the obstacle of hypoxia adaption, various precondition methods (e.g., hypoxia, heat shock, and exposure to oxidative stress) were used to accommodate the cells in vitro [11]. Hypoxia was a feasible and potential way to adapt cells before they were transplanted [12]. Exposure to a sub-lethal hypoxic would signi cantly increase the tolerance and regenerative properties of stem/progenitor cells, resulting in marked protective effects against insults in the ischemic attack [13].

The aim
To establish a conducive vascular environment, scientists developed different co-transplantation systems that interacts synergistically to form stable vessels [14]. Human umbilical cord blood mononuclear cells (UCMNCs) have generated signi cant attention in regenerative medicine for their e cacy in treating ischemic diseases [15,16]. And UCMNCs have been used with different types of stem cells in animal models [17]. Therefore, we hypothesized that co-transplantation of ASCs with UCMNCs would raise the survival e cacy for the damaged tissue and seeded stem cells.

Ethical approval
All the animal experiments were performed according to the Federation for Laboratory Animal Science Association's guidelines and approved by the Animal Care Committee of the PLA General Hospital (E2018-06-06).All experiments were carried out in compliance with the Helsinki Declaration. ASCs were obtained from a healthy man who underwent a plastic abdominal surgery, and CBMNCs were isolated from cord blood of a normally delivered female baby. Both of them were obtained from the donors with informed consent according to the institutional guidelines under the approved protocol. The cord blood was collected and processed with the approval of the 'Chinese PLA General Hospital Institutional Review Board' (L2018-12-03) and manufactured to mononuclear cells according to our good manufacturing practice (GMP) process in the Human Cell Therapy Laboratory, Chinese PLA General Hospital, China.

Isolation and Identi cation of Human ASCs
Subcutaneous abdominal adipose tissues were obtained from a healthy male who underwent a liposuction surgery. Adipose tissue was washed in phosphate-buffered saline (PBS) and minced, followed by digestion in 5 ml of type I Collagenase (1 mg/ml in 1% bovine serum albumin(BSA)/Hank's balanced saline solution; Life Technologies Japan) for 40 minutes at 37°C using a gentle MACS Dissociator (MiltenyiBiotec K.K., Tokyo, Japan) according to the manufacturer's instructions. The digested tissue was ltered through a 40-μm cell strainer (BD Falcon, Tokyo, Japan) and centrifuged at 450g for 10 minutes.
ASCs were identi ed with owcytometry analysis by incubation with primary antibodies for 40 min at 4°C in phosphate-buffered saline (PBS) supplemented with 2% FBS and 2mM ethylenediaminetetraacetic acid (EDTA). The following direct conjugated antibodies (BD Pharmingen™) were used: anti-human (PE-CD34, PerCP-CD45, PE-CD90, APC-CD44, PE-CD73,and PE-CD105). All staining was controlled with appropriate isotope control antibodies. Analysis was performed on a SORP LSRII (Becton Dickinson) equipped with ve lasers and data were collected with FACS DIVA software. Analysis was performed using FlowJo™ 10.0.8 (Treestar, Ashland, OR). All measurements were performed with three biological replicates.
Linage differentiation tests for osteogenesis, adipogenesis and chondrogenesis was performed as described before [18,19]. Osteogenic differentiation was evaluated by cellular alkaline phosphatase (ALP) activity (Alkaline Phosphatase kit, 86R; Sigma-Aldrich). Adipogenesis was con rmed by Oil red O staining of intracellular lipids, and chondrogenesis was con rmed by Alcian blue staining [19].

Hypoxia Precondition of ASCs
Hypoxic treatment protocol was referred as previously reported with some modi cation [20]. A total of 3 x 10 6 ASCs (P 3 ) were seeded on a T75 ask and cultured at 2% O 2 (hypoxia) in a ProOx-C-chamber system (Biospherix, Red eld, NY), in comparation with the ambient oxygen tension 21% O 2 (normoxia). Seventytwo hours later, cells were harvested for RNA expression analysis (Supplement Table 1).

Cord blood mononuclear cells (CBMNCs) Isolation
Isolation of CBMNCs was performed as reported before [21]. In brief, 35ml of fresh cord blood was collected from a healthy woman with an uncomplicated delivery. The blood was mixed with 7ml (1:5) Hetastarch solution (HetaSep™, STEM CELL TECH, US), and incubated at 37℃ for 30 min. Then, the cells in the oating layer were transferred to another tube, mixed with normal saline (NS) to 50ml, and centrifuged for 10min (1500rpm). After having been washed for 2 times, the cord blood cells were mixed with a 5ml lymphocyte separation medium (50494 LSM ® , Cappel TM , Shanghai), and centrifuged for 20min (1500rpm). The thin layer of mononuclear cells was extracted carefully and washed 2 times. Cell viability was evaluated with a trypan blue exclusion test, and cell concentration was brought to 1.5-2.5×10 6 cells/ml and used within 30 min.

Measurement of Heart Function
Echocardiography (Vevo770; Visualsonics, Toronto, ON, Canada) was performed at 0d and 30d after myocardial ischemia. Rats were anesthetized with 4% chloral hydrate (40 mg/kg, intraperitoneally), and imaged in the supine position at the fourth and fth intercostal space with a 710B transducer. Both 2D and M-mode images were used for measurements, and images were later analyzed by a trained blind reader using the cardiac analysis software (VisualSonics, version 2.2.3). The following variables are measured: ejection fraction (EF), fractional shortening (FS), stroke volume (SV), heart rate (HR), and left ventricle posterior wall thickening (LVPW) were measured.

Infarct Size Measurement
All the hearts were harvested at 30d and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA Inc, Torrance, Calif). The infarct and peri-infarct regions were cut into three transverse sections then and stained by Masson trichrome and hematoxylin-eosin (HE). The stained sections were measured and calculated for the average ratio of brosis area (blue) to the entire LV area (percentage of brosis area), and the average ratio of the reduced LV wall thickness in the scarred area to the intact LV wall thickness from three different sites in each wall (LV wall thinning %). For each slice, 10 randomly selected elds were captured (× 100) and images were digitized and analysed with a digital image analyser (MIQAS, Qiuwei Co, China).

Living ASCs Cell Count and Vessel Count in Necrotic Area
To count the living ASCs in heart sections, we used anti-human SRY antibody (Cat#MA5-17181, 1:200, Invitrogen, US) to trace the ASCs cells (ASCs used were only from human male) [23]. Six elds per heart section were randomly chosen and photographed under 40× magni cation with a uorescent microscope, and live cells (cells/mm 2 ) were counted in every tenth heart section across the entire region of interest.
For vascular counting, we used anti-human CD31 (Cat#ab32457, 1:200, Abcam, Shanghai) to stain the newly formed capillaries. The number of capillaries was counted under a light microscope (magni cation × 250, OLYMPUS BH2, Japan) for 10 random elds in each transverse slice and presented as the mean number of blood vessels per unit area (number/mm 2 ). Both of these performances were repeated in 8 separate sections per heart. Two independent observers were blinded to the identity of the tissues.

Statistical Analysis
Statistics were performed using SPSS 17.0 Software. Data are expressed as Mean ± Standard Error. Statistical comparisons were made using an unpaired t-test or one-way analysis of variance followed by Bonferroni multiple comparison post hoc tests where appropriate. The results were considered statistically signi cant when P<0.05.
Differentiation tests showed that cultured ASCs (P3) have a strong potential of differentiation with osteogenesis, adiposegenesis and chondrogenesis, which were speci c characteristics for mesenchymal stem cells (Fig.1A).

Live ASCs in the infarct area
Stained with anti-human SRY antibody, live ASCs cells were only detectable in HP-ASCs group and HP-ASCs + CBMNCs group (Fig.5A). The result showed that HP-ASCs group had great numbers of live cells (37.43± 12.68cells /100 nucleus). However, the combination group (21.51± 7.82cells /100 nucleus) had a much lower live cell density than the HP-ASCs group (p < 0.05) (Fig.5C).

Discussion
Hypoxia pretreatment has been demonstrated to be an effective way to accommodate the stem cells before transplantation [11]. It is also an effective way to enhance MSCs survival in myocardial ischemiareperfusion injury [24], as well as promote angiogenesis and neurogenesis in rat ischemic brain models [25]. To understand its mechanism, we analyzed the gene expression of HP-ASCs in vitro, which showed an enriched paracrine pro les. The highly raised growth factors (EGF, VEGF, PDGF, TGF-β, FGF-2 and IGF-1) have been demonstrated to be essential for cell survival and capillary buildup [26]. we also found that HP-ASCs can facilitate the reparation of the ischemic area by potentiating the chemotactic effects, based upon the evidence of up regulation of migration (CXCL12) and adhesion (ICAM-1and ICAM-2), which had been reported in previous studies [24].These ndings were in according with the previous studies which demonstrated that preconditioned stem/progenitors cells increased cell survival, enhanced paracrine effects and improved homing to the lesion site [13]. Also, these preconditioned cells helped to suppress in ammatory factors and immune responses after transplantation [11]. Some studies reported that ASCs primed with hypoxia and in ammation could enhance cardiomyocyte proliferation rate [12]. However, we did not nd evidence for myocardial differentiation in HP-ASCs in vitro (negative with myocardiocyte markers: α-actin, MYH6, NKX2.5, CX43, GATA-4) (data not shown). This inconformity was probably resulted from the different pretreatment conditions and culturing periods.
Combined transplantation has been explored for treatment in the past few years, but it still remains paradoxical because not all of the combinations would work [15,16,27]. Studies have reported that some stem/progenitor cells would improve the e cacy of MSCs/ASCs when they were co-transplantated. For example, endothelial progenitor cells (EPCs), potentially proangiogenic cells, would promote vascular buildup when combined with MSCs [28]. However, in a meta-analysis, no signi cant difference in angiogenesis was found in AMI rats, comparing the EPCs groups and MSCs+ EPCs groups, even though the latter had a better improvement in the cardiac function [17]. Different from the previous studies, we observed enhanced angiogenesis in the combination group, but not nding advantages of cotransplantation in improving cardiac function or reducing myocardial brosis, as well as elongating ASCs survival.
There might be a few reasons for the failure of the co-transplantation. First, CBMNCs were a mixture of stem/progenitor cells, which consists of endothelial progenitor cells (EPCs) [29], hematopoietic stem/ progenitor cells (HSPCs) [30], and endothelial colony-forming cells (ECFCs) [14]. Though some components can work synergistically with ASCs, while others may not [27]. Further studies are still needed to identify the fractions that impede the cell-cell interactions.
The second reason for the impediment of the combined effect was attributed to heterogeneity, which resulted from various factors, such as differences in the amounts and ratios of these two types of cells, organization source, measurement time and so on [17]. For instance, cell ratios may play an important role for determining the ultimate result in reparation, as these two types of cells may have a strong competition for oxygen and nutrition. In a previous study, scientists screened a serial of ratios between HSCs and MSCs (1:0, 1:1, 1:2, 1:4, 1:8 and 1:16), which xed an optimal ratio of 1:8-1:16 to regain the hemopoietic function in NOD/SCID mice [31]. However, people tend to use 1:1 ratio with different dosage to treat AMI, which showed various e ciency [17]. Therefore, further studies are still needed to explore the optimal conditions for co-transplantation.
Another reason for the impediment of the combined effect was that the growth factors secreted by ASCs might be deployed by CBMNCs for their survival and new vessels formation, rather than promoting ASCs' survivals. It has been demonstrated by many studies, as well as by this study, that HP-ASCs had an enriched paracrine functions before they were transplanted [26,32,33]. ASCs would support other types of cells (e.g. HSPCs) for their viability and proliferation, through direct/ paracrine cell-to-cell interactions [34,35]. In addition, ASCs can also upregulate the adhesion molecule and extracellular matrix genes in HSPCs, which was good for the latter to anchor and survive in the local area [36].All aforementioned evidence demonstrated that the paracrine factors that secreted by ASCs might be used by CBMNCs for survival and function when they are co-transplanted simultaneously. So, we need to nd new strategies to improve the treatment e cacy. A plausible method is that transplanting the different type of cells in a sequential way, i.e. transplanting CBMNCs before ASCs, so that new vessels would support the later to survive. However, the intervals need to be determined, which is neither too close to in uence individual cell's survival, nor too far to lower the treatment e ciency.

Limitations
However, we still have some limitations with our study. First, we should select the effective component that will work with ASCs synergistically by a co-culture system. Second, we need to explore the optimal intervals for successive transplantation, in order to enable the vascular buildup before the injection of ASCs. Third, we should also investigate the cross-talk between the implanted cells and the host cells. In the future studies, we would trace different types of cells with different labeled colors and carry out the experiment with organ-on-chip models.

Conclusion
In all, HP-ASCs were effective in improving cardiac function and reducing the size of infarcted myocardium in rats with AMI. When combined with CBMNCs, there would be more increase in angiogenesis, but no signi cant promotion for ASCs survival.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
All the authors were consent for publication.

Data Availability Statement
The data that support the ndings of this study are available from the corresponding authors upon request.