Pluripotin facilitates the expansion of hematopoietic stem cells, but restricts the growth of fibroblasts and the proliferation of mesenchymal stem cells from the bone marrow


 Abstract

Hematopoietic stem/progenitor cell (HSPC) quiescence modulators play a key role in the modulation of HSPC numbers. Targeting HSPC modulators with small molecules could contribute to the cell cycle entry and expansion of HSPC. To this end, we have investigated the effect of two small molecules Pluripotin and CHIR-99021 on HSPC expansion. Both Pluripotin and CHIR-99021 have been shown to result in a 3-fold rise in the murine pool of HSPC in a dose-dependent compartment after 7 days of treatments. In addition, we investigated the influence of Pluripotin treatment on the ex vivo expansion of human umbilical cord blood and bone marrow mononuclear cells. In specific, Pluripotin treatment increased human CD34 + and ALDHbr HSPC content up to 3-fold compared to control. In addition, human CD133 + HSPC cell content was increased to 5-fold following treatment with Pluripotin. Intriguingly, we find that Pluripotin treatment inversely changes bone marrow-derived mesenchymal stem cell (MSC) proliferation and lowers fibroblast growth although there is no effect on adipose-derived MSCs. CHIR-99021 treatment did not have any effect in proliferation of MSCs or fibroblasts. In conclusion, Pluripotin-induced stem cell expansion is unique to HSPCs that can be used to expand HSPCs and reduce unwanted fibroblast or MSC growth in the primary ex vivo cultures.


Introduction
The most de ning features of HSCs are their ability to regenerate and differentiate into multiple cell types. Thanks to these abilities, HSCs can transform into all types of blood cells, while at the same time maintaining the regeneration capacity necessary for the body's future hematopoietic activity. HSCs are frequently used in HSC transplants for the treatment of hematological disorders due to their self-renewal abilities. However, even if there are suitable donors for these transplants, the number of HSCs obtained is not always su cient for an effective transplant. Therefore, studies for ex vivo HSC proliferation are needed for such therapeutic applications. It has been shown in the literature that the deletion of some HSC quiescence genes in mice leads to HSC proliferation (Yucel and Kocabas, 2017).
While HSCs form the basis of bone marrow transplantation, they also hold promise for gene therapy studies . HSC transplantation is used in the treatment of leukemia, lymphoma, some solid cancers, autoimmune diseases and genetic conditions such as Sickle Cell Anemia or Mediterranean Anemia (Rameshwar et al., 2011;Zheng et al., 2011). The success of HSC transplantation depends on nding a Human Leukocyte Antigen (HLA) compatible donor and obtaining su cient number of HSCs for engraftment from one donor. Even if HLA-compatible donors are found, the insu cient number of HSCs obtained often reduces the success rate of bone marrow transplantation. Therefore, in order to obtain su cient numbers of HSCs, an alternative method such as ex vivo reproduction of HSCs should be developed. Mouse and human HSCs can be identi ed and isolated by staining surface antigens by uorescence activated cell sorting (FACS) method, and they can be reproduced to a certain extent ex vivo by treatment with TPO, FL3, and SCF cytokines (Barančıḱ et al., 2001;Watts et al., 2011). While most of the methods for multiplying HSC depend on the use of cytokines and growth factors, the use of small molecules targeting HSC-quiescence factors is uncommon. Cytokines stimulate silenced (in the G0 phase) long-term HSCs to enter the cell cycle. It is thought that they do this by increasing self-renewal factors or by suppressing cell cycle inhibitors. On the other hand, it has been observed that using a p38 inhibitor increases ex vivo HSC proliferation Zou et al., 2012). Based on all this, HSCs can be replicated by targeting silencing factors or small molecules that improve self renewal.
The aim of this study is to determine the effect of small molecules that targets stem cell quiescence or self-renewal directly or indirectly in the ex vivo HSC expansion. To this end, we have utilized pluripotin that is known to maintain self-renewal in ESCs and CHIR-99021 that is a GSK3 inhibitor and activator of WNT pathway. We have especially tested these compounds which were not fully characterized in umbilical cord blood derived HSCs. In addition, we tested the effect of these compounds in primary AD-MSCs, BM-MSCs, broblasts along with mobilized peripheral blood HSCs, and their murine counterparts. These cell types have previously not been assessed how Pluripotin or CHIR-99021 affect stem cell expansion ex vivo.

Lineage negative cell isolation
Mouse bone marrow was extracted from the femurs and tibias of BALB/c mice (YUDETAM) following euthanasia by ushing the bone marrow space with ice cold dulbecco's phosphate-buffered saline using a syringe and a 26G needle (DPBS, Invitrogen, Gibco, UK, cat no.14190250). Homogenized collecting marrow with 5ml syringe and lter the cell suspension through 70 um cell strainer (BD Pharmingen, 352350) then centrifuged cells and remove the supernatant. Resuspended cells into FC block, Lin Cocktail (BD, 560492) and Cell Staining Buffer, then washed the cells with Imag Buffer. The labelled cells were incubated sequentially with the following reagents, each step being for 15 minutes on ice. For negative selection labelled cells by using biotinylated antibody plus Streptavidin and after 30 min incubation on ice, the labelled cells are resuspended in 1X BD IMag buffer. The labelled cells were transferred into ow tubes and tubes and placed into BD IMagnet. In this way labelled cells are attracted to the magnet and positive cells stick on the wall of ow tubes. Supernatants which contain desired cells, were collected from the ow tube without removing inside BD IMagnet. The isolated cells were seeded on 96 well-plate in expansion medium at a density of 30,000 cells per well for ow cytometry analysis as we have done previously .

Flow cytometry analysis of murine HSPCs
After 7 days of the treatment with the small molecules which are CHIR-99021, Pluripotin and DMSO as a control, lineage negative cells were labelled with Fc Blocker into 1X PBS then surface marker staining for APC-lineage cocktail, PE-C-kit, PE-Cy7-Sca-1 and FITC-CD34 (BD StemFlow Cat No 560492) according to the manufacturer's manual (Stemcell Technologies). The expressions of CD34 Sca-1 and c-Kit, surface markers in the labelled cells were analysed by ow cytometry (BD FACSARIA III) (Turan et al., 2020).

Bone marrow derived mesenchymal stem cell isolation
Following a procedure modi ed by Soleimani and Nadri, bone marrow mesenchymal stem cells were isolated (Nadri et al., 2008). Isolated bone marrow cells were seeded with 15 percent (v/v) FBS and 1 percent (v/v) PSA at 30x10 6 cells on T-75cm2 asks in DMEM. Non-adherent cells were discarded the next day and the medium was altered every 3-4 days. The cultured cells in passage 1 were treated with DMSO, CHIR-99021 and Pluripotin at 0.1 μM, 1 μM and 10 μM.

Umbilical cord blood mononuclear cell isolation and treatment
Umbilical cord blood cells were harvested with the permission of the parent through Onkim Stem Cell Technologies. By Ficoll-Paque (HistopaqueTM, Sigma, CatNo.10831) density gradient centrifugation (1,083g/ml), UCB and BM mononuclear cells were isolated and plated in 96 well-plate in expansion medium for ow cytometry analysis at 10,000 cells per well. The expansion medium consists of Serum-Free Expansion Medium (StemSpanTM) 1 percent PSA and human cytokine cocktail (StemSpanTM CC100). The plated cells were treated with small molecules of CHIR-99021 and Pluripotin at concentrations of 0.1 μM, 1 μM and 10 μM and DMSO as a control. Dimethyl sulfoxide (0.5 percent) was treated with the cells used as control (Calbiochem, 317275) .

Cell Cycle Analysis
To determine that whether the LSK and CD34+ cells re-entry to cell cycle or not, murine LSK (Lin -Sca1 + Ckit + ) cells from mouse lineage (-) cell population was separated by ow cytometry (FACSARIA III, BD Biosciences, Cat.No. 23-11539-00). The separated cells were seeded on the proper expansion medium at 5000 cells per well in 96 well-plate and treated with the effective doses of CHIR-99021 and Pluripotin. The cells were stained with Hoechst 33342 (10 μg/ml) and Pyronin Y (100 μg/ml) after 4 days of therapy and analyzed by ow cytometry, as we had previously done (Aksoz et al., 2019).

AD-MSC isolation and ow cytometric analysis
Adipose tissue was derived through liposuction surgery. A 500 ml container of 60 ml of adipose tissue was put in and the same volume of collagenase solution was applied. At 37°C, the tissue was digested by constant shaking for 1 hour. At RT, the digested tissue was centrifuged for 7 min at 2500 rpm. The adipocytes on the supernatant were discarded and 2 ml of erythrocyte lysis buffer was re-suspended with the pellet. The cell suspension was nished with the erythrocyte lysis buffer at 50 ml and incubated by constant shaking at 37 °C for 10 minutes. The pellet was re-suspended in a 6 ml expansion medium then ltered through 100 µm cell strainer. Cells were seeded as 10 6 cells/150 cm 2 on tissue culture polystyrene asks. After 24 hour cultured, the medium was refreshed. For treatment, 10.000 cells in passage 1 per well were seeded on 96-well plates and treated with CHIR-99021 and Pluripotin. After 7 days of treatment, cells were stained with anti-human CD73-APC, CD90-FITC, CD105-PerCP/CY5.5 and CD45-PE and analyzed by ow cytometry as we have done previously (Aksoz et al., 2019).

Cell viability analysis
Human AD-MSC and murine BM-MSC were seeded on 96 well plates at a density of 2000 cells per well.
Cells were grown in DMEM supplemented with 10% FBS, 1% (PSA) Antibiotic-Antimycotic (100X). One day after, small molecules were added into each well with 0.1 μM, 1 μM and 10 μM concentrations of CHIR-99021 and Pluripotin for treatment. Human dermal broblasts (HDFs) were seeded at a density of 5000 cells per well on 96 well plates and treated with 0.1 μM, 1 μM and 10 μM concentrations of CHIR-99021 and Pluripotin. The reagent was diluted with culture medium within 1:10 after 72 hours of treatment (Cell Proliferation Reagent WST-1, Roche, Cat No. 11644807001). The cells were treated with the reagent and incubated at a cell culture incubator and measured the absorption of the samples using the Thermo Labsystem Multiskan Spectrum microplate reader at 420-480 nm.

Human and animal experiments have been authorised by the Institutional Clinical Studies Ethical
Committee of the University of Yeditepe (#1067 and #944) and YUDHEK (#429).

Statistical analysis
The statistics have been evaluated by the Student T Test. p<0.05 was considered statistically signi cant.

Pluripotin treatment improves murine, cord blood and G-CSF mobilized peripheral blood HSPC expansion
Hematopoietic stem cells are rare cells in the bone marrow that need to be expanded if they are needed in large quantities. Mouse bone marrow was collected from the femurs and tibias of BALB/c mice. In order to improve the expansion of HPSCs, small molecules (SM) were tested alongside known growth factors and cytokines. Lin-cells separated by magnetic depletion and seeded by SFEM media which comprise basic growth factors such as SCF, TPO and FLT3-L. Cells treated with CHIR-99021 and Pluripotin small molecules and DMSO as a control immediately afterwards seeding. HPCs were analysed with ow cytometry post 7 days after treatment with different dosages of CHIR-99021 and Pluripotin small molecules. According to the ow cytometry results, the e cient dosage that increased HSPC number was 0.1 µM for CHIR-99021 and 1 µM for Pluripotin small molecules ( Figure 1A).
Following identi cation of e cient dosage, the current doses applied in LSK cells enriched for HSPCs in order to elevate HSPCs expansion. LSK cells sorted after Lin-depletion. Cells treated with CHIR-99021, Pluripotin and DMSO as a control, immediately afterwards seeding sorted cells. HPCs expansion were analysed with ow cytometry post 7 days after treatment with e cient dosages of CHIR-99021 and Pluripotin small molecules. According to the ow results which shown in Figure 1B, HSPC expansion were approximately 4 fold higher in CHIR-99021 small molecules and 18 fold higher in Pluripotin small molecules than that in DMSO group. When we compared among each other, Pluripotin enhanced 4.5 fold higher than CHIR-99021 treatment alone. In addition, both CHIR-99021 and Pluripotin treatment allowed HSC to exit from G0 and enriched in S-G2-M phases of HSPC cell cycle ( Figure 1C), without any signi cant alteration in the rate of apoptosis ( Figure 1D).
Umbilical cord blood mononuclear cells (UCB MNCs) are a rich source of HSPCs. UCB MNCs were isolated and were seeded in SFEM media which comprise human cytokine cocktails. UCB MNCs were treated with CHIR-99021, Pluripotin and DMSO as a control immediately afterwards seeding. HPSCs were analysed with ow cytometry post 7 days. According to live-cell counting by ow cytometry results, total UCB MNC content of averages of three different dosages SMs were increased by both CHIR-99021 and Pluripotin (Figure 2A). Similar to CHIR-99021 treatment, Pluripotin treatment also increased CD34+ HSPC content approximately 2 fold higher in CHIR-99021 and 3 fold higher in Pluripotin than that in DMSO group ( Figure 2B). In addition, CD133+ cells content were 5 fold higher for CHIR-99021 and 10 fold higher for Pluripotin treatment compared to that in DMSO treatment ( Figure 2C). Besides HSPCs expansion analysis, we determined ALDH (Aldehyde Dehydrogenase) enzyme activity in UCB MNC post SM treatments. Even though CHIR-99021 could not make a signi cant difference in ALDH br HSPC content, Pluripotin represented up to 3-fold increase compared to control ( Figure 2D). G-CSF mobilized human peripheral blood mononuclear cells (mPB MNCs) are another rich source of human HSPCs. We also obtained mPB MNCs and culture in SFEM media supplemented with human cytokine cocktails and treated with CHIR-99021, Pluripotin and DMSO as a control immediately afterwards seeding. HPC content was analysed with ow cytometry post 7 days after treatment with different dosages of CHIR-99021 and Pluripotin small molecules. CD34+ HSPC content was about 1.5 fold higher in CHIR-99021 and 2 fold higher in Pluripotin than that in DMSO group ( Figure 3A). In addition, CD133+ HSPC cell content was increased up to 5-fold following Pluripotin treatment in mPB MNCs ( Figure 3B). Furthermore, when we analysed CD34+CD133+ HSPC content of mPB MNCs, we have determined over 1.5 fold increase in Pluripotin treated group than that in DMSO group, while there is no visible change in CHIR-99021 treated group ( Figure 3C).

Pluripotin treatment limits bone marrow derived mesenchymal stem cell expansion
To address the effect of Pluripotin and CHIR-9902 on other stem cells, we studied bone marrow derived mesenchymal stem cells (BM-MSCs) and adipose derived mesenchymal stem cells (AD-MSCs). In addition, we tested a potential source of cell contamination in the ex vivo cultures by analysis of broblast growth using HDFs. Percentage of viable murine bone marrow mesenchymal stem cells were analysed according to WST1 absorbance post 3 days after treatment with different dosages of CHIR-99021 and Pluripotin small molecules. These results consist of an average of different dosages which are 0.1 µM, 1 µM, 10 µM and compared with DMSO ( Figure 4). Intriguingly, the results indicate that Pluripotin treatment inversely changes proliferation kinetics of BM-MSCs ( Figure 4A), as well as human broblasts ( Figure 4B) while there is no effect on AD-MSCs ( Figure 4C).
Overall these results indicate that Pluripotin and CHIR-99021 stimulate murine, human cord blood, and mobilized peripheral blood HSPC expansion ex vivo ( Figure 5). Interestingly, the negative effect of Pluripotin on MSCs and broblasts was unique that could be further studied in procedures that require the elimination or reduction of MSCs, broblasts or similar mesenchymal cell growth along with HSPC expansion procedures, and ultimately increase transplantation e ciency.

Discussion
Su cient HSCs are needed for an effective transplantation and settlement. This requires functional HSC replication in a method that is reliable for recipients. Various problems are encountered in studies for ex vivo HSC reproduction, such as loss of self-renewal ability in HSCs, increased differentiation in cells during replication, and limited knowledge of HSC regulators [1]. Cytokines such as thrombopoietin (TPO), FL3, IL3, IL6, IL11, and stem cell factor (SCF) have been shown to increase HSCs. However, problems are encountered in long-term and effective reproduction (Rameshwar et al., 2011;Watts et al., 2011;Yucel and Kocabas, 2017;Zheng et al., 2011).
There are studies showing the effects of inhibitors on ex vivo proliferation of HSC [3,8,9]. These studies include the use of StemRegenin 1 (AhR antagonist), Garcinol (nonspeci c HAT inhibitor) and Nicotinamide (SIRT1 inhibitor) to reproduce human and mouse HSCs (Boitano et al., 2010;Peled et al., 2012;Singh et al., 2011). We have recently shown that the regulation of hematopoietic factors such as Meis1 and Hif-1a in HSCs and that these factors are needed for HSC quiescence (Kocabas et al., 2012)and targeting with MEIS inhibitors could allow ex vivo HSC expansion (Turan et al., 2020).
It has become a deep new prospective for the ES-cell-renewal mechanisms since pluripotin promotes ES cell autoself-renewal independently of its exogenous activation in conventional self-renewal paths simply by inhibiting the role of the endogenous differentiation-induced proteins (Semi and Takashima, 2021).
The key to maintaining ES cells in a self-renewable state is to suppress the harmful effects of endogenic expression on the inducers of differentiation or cell death. This is also con rmed by the combination of unique chemical inhibitors, including CHIR99021 and PD03259012, also supported the derivation and long-term self-renewal of mES cells in the absence of exogenous cytokines (Ying et al., 2008). For example, the derivation of embryonic stem cell lines from refractory strains is signi cantly facilitated by pluripotin combined with leukemia inhibitory factor (Yang et al., 2009). These also provide a platform for a consistent and robust cell culture as well as for the derivation of new cell lines from di cult strains or species. A recent study showed that CHIR 99021 increases the differentiation potential of mesenchymal stem cells (Govarthanan et al., 2020). A GSK inhibitor, in combination with p38 inhibitor has been recently shown to allow HSC expansion (Li et al., 2019).
CHIR 99021 inhibits activity of GSK3. GSK3-β is highly expressed in the hematopoietic compartment ( Figure S1A). On the other hand, in embryonic stem cells, Oct4 and the small molecule inhibitor, Pluripotin, could modulate Tet2 expression ( Figure S1B) (Wu et al., 2012). They demonstrated that Pluripotin (also known as SC1) represses the expression of Tet1 and Tet2. Previous studies also showed that conditional loss of Tet2 in the hematopoietic compartment contributes to HSPC expansion and increased vivo repopulation ability (Li et al., 2011;Moran-Crusio et al., 2011;Quivoron et al., 2011). Our analysis also indicates that Tet2 is highly expressed in hematopoietic cells ( Figure S2B), its downregulation following pluripotin treatment could be associated with the phenotype that we observe in HSC expansions.
Two small pluripotin molecules and CHIR-99021 have been examined for HSC growth. The result was a growth in HSC murine pool in the dose-based manner after seven days of therapy, as demonstrated by both Pluripotin and CHIR-99021. We also examined the effect of pluripotin therapy on the ex vivo expansion of blood of the human umbilical cord and mononuclear bone marrow cells. We nd it intriguing to note that, although pluripotin treatment has no effect on adipose derivative MSC, it inversionly changes the expansion of MSC by bone marrow. Treatment with CHIR-99021 had no effect on MSC or broblast proliferation. Finally, the pluripotin induced expansion of stem cells can be used to expand HSCs in the primary ex-vivo culture and to reduce unwanted broblast or MSC.
Con icts of interest/Competing interests : FK is the founder of Meinox İlaç Teknolojileri A.Ş. All other authors declare that they have no con ict of interest. We like to thank our partners and fellow scientists who helped us in sample collections, initial observations and ethical committee approvals.
Availability of data and material: Supporting data is available upon request.
Code availability: Not applicable Authors' contributions: RDT performed experiments and wrote the article. FK designed the studies and wrote the article.