Differentiated Cells Derived from Hematopoietic Stem Cells and Their Applications in Translational Medicine.

Hematopoietic stem cells (HSCs) and their development are one of the most widely studied model systems in mammals. In adults, HSCs are predominantly found in the bone marrow, from where they maintain homeostasis. Besides bone marrow and mobilized peripheral blood, cord blood is also being used as an alternate allogenic source of transplantable HSCs. HSCs from both autologous and allogenic sources are being applied for the treatment of various conditions like blood cancers, anemia, etc. HSCs can further differentiate to mature blood cells. Differentiation process of HSCs is being extensively studied so as to obtain a large number of pure populations of various differentiated cells in vitro so that they can be taken up for clinical trials. The ability to generate sufficient quantity of clinical-grade specialized blood cells in vitro would take the field of hematology a step ahead in translational medicine.

Curcumin restores the engraftment capacity of aged hematopoietic stem cells and also reduces PD-1 expression on cytotoxic T cells.

Aging affects the functionality of hematopoietic stem cells (HSCs), and therefore, aged individuals are not preferred as donors in HSC transplantation. Such elimination leads to the restriction of donor cohort. Several efforts are being done to rejuvenate aged HSCs. Here, we show that treatment of aged mice with curcumin rejuvenates their HSCs by restoring the expression of autophagy-inducing messenger RNAs in them, and improves their engraftment capacity. Importantly, we show that curcumin is effective in rejuvenation of HSCs when administered via both, intraperitoneal as well as oral routes. Aging also affects the immune system. While elderly individuals are not immuno-deficient, they do not respond optimally to immunizations, and hence, a strategy needs to be developed to make them immunologically responsive. Programmed cell death 1 (PD-1), one of the inhibitory coreceptors, plays an important role in the regulation of autoimmunity, infectious immunity, and cancer immunity. Its expression on T cells is indicative of their exhaustion. Here, we show that curcumin reduces the frequency of PD1+ cytotoxic T cells in the spleens of aged mice. Curcumin has a proven safety profile, and hence, can be used to treat aged donors to boost the functionality of their HSCs and also to improve the immunological profile of aged individuals. These data could have implications in various other regenerative medicine protocols as well.

Establishment of human iPSC line from patient of Indian ethnicity carrying homozygous CD8/9 (+G) beta thalassemia mutation.

This study shows generation of iPSCs from peripheral blood mononuclear cells (PBMNCs) of a male patient having homozygous CD 8/9 (+G) beta thalassemia (major) mutation. Cells were nucleofected with episomal vectors containing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (dominant negative p53 mutation). Cell line exhibited presence of pluripotency markers by immunofluorescence, flow-cytometry and PCR. The plasmids were lost from cells by subsequent passages, observed by PCR. Karyotype analysis demonstrated a stable genome. The cells had capability to differentiate into three-germ lineages in vitro. This iPSC line can be used as a tool for drug design and gene therapy studies.

Transforming growth factor ß1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy.

BACKGROUND: Generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) in vitro takes about 21 days, making it unaffordable for clinical applications. Acceleration of the in vitro erythropoiesis process by using small molecules could eventually make the large-scale production of these cells commercially viable. Transforming Growth Factor ß1 (TGF-ß1) has been shown to have a dose-dependent activity on the HSCs: at high concentration it inhibits, whereas at low concentration it stimulates the HSCs growth. At high concentration, it also inhibits erythropoiesis but accelerates terminal erythroid differentiation of cell lines and erythroid progenitors. Here we examined whether the use of low concentration of TGF-ß1 would be beneficial for increasing RBC production by stimulating HSC growth and also supporting erythroid differentiation. Such a strategy could make RBC production in vitro more efficient and cost-effective for clinical applications. METHODS: HSCs isolated from Apheresis samples were differentiated into mature RBCs by the sequential addition of specific combinations of growth factors for 21 days. In the control set, only EPO (3 IU/ml) was added whereas, in the test set, TGF-ß1 at a concentration of 10 pg/ml was added along with EPO (3 IU/ml) from day 0. RESULTS: We found that a low concentration of TGF-ß1 has no inhibitory effect on the proliferation of the early stages of erythropoiesis. Additionally, it significantly accelerates terminal stages of erythroid differentiation by promoting BNIP3L/NIX-mediated mitophagy. CONCLUSIONS: Incorporation of TGF-ß1 at 10 pg/ml concentration in the differentiation medium accelerates the in vitro erythropoiesis process by 3 days. This finding could have potential applications in transfusion medicine.

Neutrophils generated in vitro from hematopoietic stem cells isolated from apheresis samples and umbilical cord blood form neutrophil extracellular traps.

Neutrophils release neutrophil extracellular traps (NET) comprising of decondensed chromatin that immobilizes and kills pathogens. In vitro generation of neutrophils on a large scale from hematopoietic stem cells (HSCs) may be a useful strategy for treating neutropenic patients in future, though it is not in clinical practice yet. Microbial infections lead to major cause of morbidity and mortality in these patients. Despite the importance of NET in preventing infection, efficacy of in vitro-generated neutrophils from HSCs to form NET is not tested. We show that functional neutrophils could be generated in vitro from HSCs/MNCs isolated from umbilical cord blood (UCB) and apheresis-derived peripheral blood (APBL). Neutrophils generated from UCB showed properties comparable to those isolated from peripheral blood. We also show that isolation of HSCs is not absolutely essential for in vitro neutrophil generation. Further, we show that neutrophils generated from HSCs express PADI4 enzyme and their NET-forming ability is comparable to peripheral blood neutrophils. Taken together, our data show that fully functional neutrophils can be generated in vitro from HSCs. NET-forming ability of in vitro-generated neutrophils is an important parameter to determine their functionality and thus, should be studied along with other standard functional assays.

Catalase incorporation in freezing mixture leads to improved recovery of cryopreserved iPSC lines.

Among the various types of stem cells, induced pluripotent stem cells (iPSCs) have gained much attention due to their pluripotent nature. iPSCs help us to understand the processes that regulate pluripotency and specialization. However, in order to use them in various applications in regenerative medicine, their efficient cryopreservation and recovery after the freezing injury is critical. Here we have used an antioxidant catalase, as an additive to the conventional freezing mixture containing 50% FBS and 10% DMSO. The hiPSCs were frozen as aggregates by using a programmable freezer and then stored in liquid nitrogen at -196 °C. It was seen that catalase improved the revival efficiency by reducing the late apoptotic populations and increasing the live cell fraction. Catalase also retained the pluripotent nature of iPSCs in a better way post revival. This improvement could be attributed to reduction of total ROS and apoptosis, which are the two main factors that cause damage during freezing. Our data suggest that catalase could be a useful additive while freezing hiPSCs.

Flow Cytometry and Cell Sorting Using Hematopoietic Progenitor Cells.

Flow cytometry is a widely used laser-based technology for rapid analysis of the expression of cell surface antigens and intracellular molecules in various cell types including hematopoietic stem/progenitor cells (HSPCs). Multiparametric analysis of individual cells within a short time frame makes this tool attractive and indispensable in the field of stem cell research. This is accomplished by harnessing the specific light scattering ability of the cell type, which determines its size and internal complexity. In addition, use of fluorescently conjugated antibodies allows the detection of a specific surface or intracellular antigen present at that particular stage. Fluorescent Activated Cell Sorting (FACS) is used to separate a subset of cells from a heterogeneous cell population based on fluorescent labeling. Here we describe the general principles of flow cytometry and detailed methods for the isolation of HSPCs using flow cytometry as a tool.

Autologous Hematopoietic Stem Cells Are a Preferred Source to Generate Dendritic Cells for Immunotherapy in Multiple Myeloma Patients.

In multiple myeloma (MM), dendritic cells (DCs), and their precursors are prone to malignant cell-mediated regulation of function leading to low efficacy of DC vaccine. DCs taken directly from MM patient's body or derived from monocytes are fewer in numbers and are also dysfunctional. Here, we investigated the functionality of Hematopoietic stem cell-derived DCs (SC-DCs) from MM patients. Mature-MM-SC-DCs showed all essential functions like antigen uptake, allogenic T cells simulation and migration comparable to those derived from healthy donor (HD) samples. A comparison of Mo-DCs and SC-DCs obtained from the same MM patients' samples revealed that the expression of IL-6 was higher in the precursors of Mo-DCs leading to their impaired migration. In addition, expression of CCR7 which is responsible for DCs migration was found to be lower in MM-Mo-DCs. The chromatin permissiveness as observed by H3K4me3 histone modification at the Ccr7 promoter in MM-Mo-DCs was significantly lower than those in MM-SC-DCs. Levels of Zbtb46- a hall mark DC transcription factor mRNA was also found to be reduced in MM-Mo-DCs. Cytotoxic T cells generated from MM-SC-DCs from autologous naïve T cells exhibited reduced antitumor activity because the T cells were exhausted. Blocking of CTLA-4 on autologous T cells could partially restore T cell proliferation and activation. Thus, a combination of MM-SC-DC vaccine and anti-CTLA-4 antibody may serve as a better candidate for immunotherapy of MM. This study has implications in increasing the efficacy of cancer immunotherapy in MM.

Improved neural differentiation of normal and abnormal induced pluripotent stem cell lines in the presence of valproic acid.

During the generation of induced pluripotent stem cell (iPSC) lines from cord blood CD34+ cells, a line having complete trisomy of Chromosome 1 and deletion of q23 to qTer of Chromosome 11 was accidentally developed in our lab. The abnormality was consistently detected even at higher passages. These chromosomal anomalies are known to manifest neurological developmental defects. In order to examine if such defects occur during in vitro differentiation of the cell line, we set up a protocol for neural differentiation. Valproic acid (VPA) was earlier reported by us to enhance neural differentiation of placental mesenchymal stem cells. Here, we induced normal and abnormal iPSC lines to neural lineage with/without VPA. Neural differentiation was observed in all four sets, but for both the iPSCs lines, VPA sets performed better. The characteristics tested were morphology, neural filament length, detection of neural markers, and electrophysiology. In summary, the karyotypically abnormal line exhibited efficient neural differentiation. This iPSC line may serve as a useful tool to study abnormalities associated with trisomy 1 and deletion of q23 to qTer of Chromosome 11.

Chimeric feeders of mesenchymal stromal cells and stromal cells modified with constitutively active AKT expand hematopoietic stem cells.

Aim: To examine whether AKT-modified stromal cells expand human CD34+ hematopoietic stem cells (HSCs). Methods: Coculture, in vitro functional assays, immuno-fluorescence microscopy, flow cytometry. Results: M2-10B4 stromal cells (M2) modified with AKT1 (M2-AKT) expanded primitive CD34+38- HSCs, but affected their functionality. A chimeric feeder layer comprising naive human bone marrow-derived mesenchymal stromal cells and M2-AKT not only overcame the negative effects of M2-AKT, but, unexpectedly, also gave a synergistic effect on the growth and functionality of the HSCs. Conditioned medium of bone marrow stromal cells worked as effectively, but cell-cell contact between HSCs and M2-AKT cells was necessary for the synergistic effect of M2-AKT and bone marrow-derived mesenchymal stromal cells or their CM. Conclusion: Chimeric feeders expand HSCs.

Development and characterization of human iPSC line NCCSi004-A from umbilical cord blood (UCB) derived CD34+cells obtained from donor belonging to Indian ethnic population.

Here we report the reprogramming of CD34+ cells obtained from UCB of a healthy donor female child belonging to the Indian ethnic population. These CD34+cells were subjected to nucleofection for delivery of episomal vectors expressing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (negative mutation in p53). The iPSC colonies expressed pluripotency markers as detected by PCR, immunofluorescence and flow-cytometry. The removal of plasmid was confirmed by its absence in cells at higher passages. Karyotype analysis revealed a stable genome. The property of in vitro differentiation to tri-lineage was confirmed by expression of markers by immunofluorescence.

Regulation of differentiation of MEG01 to megakaryocytes and platelet-like particles by Valproic acid through Notch3 mediated actin polymerization.

Valproic acid (VPA) is one of the HDAC inhibitors used for the treatment of neurological disorders and hematological malignancies. Its role in self-renewal and proliferation of hematopoietic stem cells (HSCs) is well studied, but little is known about its involvement in regulating megakaryopoiesis and thrombopoiesis. In this study, we evaluated the role of VPA in megakaryopoiesis by using MEG-01, a megakaryoblast cell line. Our results show that VPA treatment differentiates MEG-01 cells to megakaryocytes (MK) and platelet-like particles. It was confirmed by augmented expression of MK and PLT-specific markers, higher ploidy, and PLT functionality. We assessed the molecular events underlying megakaryopoiesis. In the present study, we found an upregulation of Notch3 and its downstream target PDGFR-ß upon VPA treatment. The direct role of Notch3 in megakaryopoiesis has not yet been studied. PDGFR-ß is known to control actin organization during vascular smooth muscle cell differentiation. The actin cytoskeleton plays important role during proplatelet and PLT formation. We found an upregulation of Rac/Cdc42 GTPase and its downstream effectors that are the key players during actin polymerization events. We speculate that VPA induces PLT formation through Notch-3 signaling that in turn modulates actin polymerization that is one of the crucial steps necessary for thrombopoiesis. These studies were also confirmed with knockdown of Notch3 in MEG01 by using ShRNA approach as well as with apheresis-derived CD34+ cells. Altogether, these findings provide an evidence for a novel role of Notch3 in regulating platelet formation.

Microvesicles Secreted by Nitric Oxide-Primed Mesenchymal Stromal Cells Boost the Engraftment Potential of Hematopoietic Stem Cells.

Patients with leukemia, lymphoma, severe aplastic anemia, etc. are frequently the targets of bone marrow transplantation, the success of which critically depends on efficient engraftment by transplanted hematopoietic cells (HSCs). Ex vivo manipulation of HSCs to improve their engraftment ability becomes necessary when the number or quality of donor HSCs is a limiting factor. Due to their hematopoiesis-supportive ability, bone marrow-derived mesenchymal stromal cells (MSCs) have been traditionally used as feeder layers for ex vivo expansion of HSCs. MSCs form a special HSC-niche in vivo, implying that signaling mechanisms operative in them would affect HSC fate. We have recently demonstrated that AKT signaling prevailing in the MSCs affect the HSC functionality. Here we show that MSCs primed with nitric oxide donor, Sodium nitroprusside (SNP), significantly boost the engraftment potential of the HSCs co-cultured with them via intercellular transfer of microvesicles (MVs) harboring mRNAs encoding HSC-supportive genes. Our data suggest that these MVs could be used as HSC-priming agents to improve transplantation efficacy. Since both, nitric oxide donors and MSCs are already in clinical use; their application in clinical settings may be relatively straight forward. This approach could also be applied in regenerative medicine protocols. Stem Cells 2019;37:128-138.

Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation.

BACKGROUND: Dendritic cell (DC) vaccination involves administration of multiple doses. Cryopreservation of tumor antigen-pulsed DCs can provide a ready to use vaccine source and eliminate the need of frequent withdrawal of the patient's blood for vaccine preparation. The aim of this study was to assess the effect of addition of trehalose in the freezing medium on the recovery of DCs after cryopreservation. STUDY DESIGN AND METHODS: DCs were generated from mononuclear cells from apheresis samples of healthy donors. For long-term storage of 6 months, cells were frozen with a rate-controlled programmable freezer and stored in liquid nitrogen. For short-term storage of 1 month, cells were frozen and stored at -80°C. DCs frozen with Iscove's Modified Dulbecco's Medium + 10% dimethyl sulfoxide + 20% fetal bovine serum served as the control group, while the test group was additionally supplemented with 50 µg/mL of trehalose. After revival of control and test DCs, they were assessed for viability, morphology, phenotype, and functions. RESULTS: The addition of trehalose to the conventional freezing medium helped to preserve the viability and functionality of DCs better than dimethyl sulfoxide alone in both long- and short-term cryopreservation. Trehalose also protected the mitochondrial membrane potential and cytoskeleton integrity of DCs, which are necessary for their functionality. Mediators of the intrinsic apoptotic pathway like Caspase-9 and Bim-1 were found to be low in the test. CONCLUSION: Supplementation of conventional freezing medium with trehalose results in better quality of DCs revived after cryopreservation. This finding could help improve DC vaccine preparation for cancer immunotherapy.

Compromised functionality of monocyte-derived dendritic cells in multiple myeloma patients may limit their use in cancer immunotherapy.

Dendritic cells (DCs) have the potential to elicit long-lasting anti-tumour immune responses. Most of the clinical trials of anti-cancer DC vaccines are based on monocyte-derived DCs (Mo-DCs). However, their outcomes have shown limited promise especially in multiple myeloma (MM) patients. Here, we investigated whether in vitro generated Mo-DCs from MM patients (MM-DCs) possess impaired functionality, thus contributing to the limited success of DC vaccines. We generated MM-DCs and compared them with DCs from healthy donors (HD-DCs). The yield of DCs in MM was 3.5 fold lower than in HD sets. However morphology, phenotype, antigen uptake and allo-T cell stimulation were comparable. Migration and secretion of IL12p70 and IFN-γ (in DC-T cell co-cultures) were significantly reduced in MM-DCs. Thus, MM-DCs were compromised in functionality. This impairment could be attributed to autocrine secretion of IL6 by MM-monocytes and activation of their P38 MAPK pathway. This indicates a need to look for alternative sources of DCs.

Derivation of human iPSC line NCCSi002-A from umbilical cord blood (UCB) CD34+cells of donor from Indian ethnicity.

We discuss the reprogramming of CD34+ cells isolated from UCB of a healthy female child of Indian ethnicity. The CD34+cells were nucleofected using episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). The colonies were stained for alkaline phosphatase and evaluated for pluripotency marker expression by PCR, immunofluorescence and flow-cytometry. The safety of cells was confirmed by absence of plasmid in subsequent passages by PCR. G-banded karyotype demonstrated a stable genome. The ability of tri-lineage differentiation was confirmed by specific marker expression by immunofluorescence invitro and teratoma formation invivo.

Establishment of human iPSC line NCCSi003-A from CD34+cells of peripheral blood collected during apheresis of healthy donor from Indian ethnicity.

We present generation of iPSCs from CD34+ cells isolated from peripheral blood, collected during apheresis of a healthy female individual. We nucleofected the CD34+cells by episomal vectors containing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (dominant negative mutation in p53). The resultant colonies showed cobble-stone appearance and stained positive for alkaline phosphatase. The colonies demonstrated presence of pluripotency markers by immunofluorescence, flow-cytometry and PCR. The plasmids were lost from cells subsequently during passages as assessed by PCR. Karyotype analysis demonstrated a stable genome. The cells had capability to differentiate to cells from all three-germ lineages in vitro.

Intercellular Transfer of Microvesicles from Young Mesenchymal Stromal Cells Rejuvenates Aged Murine Hematopoietic Stem Cells.

Donor age is one of the major concerns in bone marrow transplantation, as the aged hematopoietic stem cells (HSCs) fail to engraft efficiently. Here, using murine system, we show that a brief interaction of aged HSCs with young mesenchymal stromal cells (MSCs) rejuvenates them and restores their functionality via inter-cellular transfer of microvesicles (MVs) containing autophagy-related mRNAs. Importantly, we show that MSCs gain activated AKT signaling as a function of aging. Activated AKT reduces the levels of autophagy-related mRNAs in their MVs, and partitions miR-17 and miR-34a into their exosomes, which upon transfer into HSCs downregulate their autophagy-inducing mRNAs. Our data identify previously unknown mechanisms operative in the niche-mediated aging of HSCs. Inhibition of AKT in aged MSCs increases the levels of autophagy-related mRNAs in their MVs and reduces the levels of miR-17 and miR-34a in their exosomes. Interestingly, transplantation experiments showed that the rejuvenating power of these "rescued" MVs is even better than that of the young MVs. We demonstrate that such ex vivo rejuvenation of aged HSCs could expand donor cohort and improve transplantation efficacy. Stem Cells 2018;36:420-433.

Data on the effect of oral feeding of Arachidonic acid or Docosahexanoic acid on haematopoiesis in mice.

Stem cells have peculiar property to self-renew and differentiate. It is important to control their fate in safe and effective ways for their therapeutic use. The mediators of essential polyunsaturated fatty acids (PUFAs) namely Arachidonic acid (AA) and Docosahexanoic acid (DHA) are known to play a role in haematopoiesis via various metabolic pathways [1]. However the direct effect of purified AA or DHA on haematopoiesis has not been well investigated yet. We have reported that oral administration of PUFAs enhanced haematopoiesis in mice [2]. Signaling Leukocyte Antigen Molecule (SLAM) (CD48-CD150+) phenotype consists of pure population of haematopoietic stem cells (HSCs). Herein we observed higher percentage of SLAM (CD48-CD150+) phenotype in the bone marrow (BM) cells of mice fed with AA or DHA compared to PBS fed control mice. Data from engraftment study depicts that BM from AA/DHA-fed mice showed higher absolute number of donor cells in recipient mice compared to control. The enhanced hematopoiesis observed in AA/DHA-fed mice was returned to normal when the mice were kept on normal diet for six weeks (after ten days of oral feeding). We confirmed GCMS (Gas Chromatography-Mass Spectroscopy) retention times of AA and DHA by co-injecting fatty acid extract from AA or DHA fed mice with purified AA or DHA standards respectively. Representative flow cytometry profile of Lin-Sca-1+c-kit+(LSK) cells showed higher expression of CXCR4 protein and ligands of Wnt, Notch1 signaling in BM of AA/DHA-fed mice.