Cancer stem cells (CSC) maintain both undifferentiated self-renewing CSCs and differentiated, non-self-renewing non-CSCs through cellular division. However, molecular mechanisms that maintain self-renewal in CSCs versus non-CSCs are not yet clear. Here, we report that in a transgenic mouse model of MYC-induced T-cell leukemia, MYC, maintains self-renewal in Sca1 þ CSCs versus Sca-1 À non-CSCs. MYC preferentially bound to the promoter and activated hypoxia-inducible factor-2a (HIF2a) in Sca-1 þ cells only. Furthermore, the reprogramming factors, Nanog and Sox2, facilitated MYC regulation of HIF2a in Sca-1 þ versus Sca-1 À cells. Reduced expression of HIF2a inhibited the self-renewal of Sca-1 þ cells; this effect was blocked through suppression of ROS by N-acetyl cysteine or the knockdown of p53, Nanog, or Sox2. Similar results were seen in ABCG2 þ CSCs versus ABCG2 À non-CSCs from primary human T-cell lymphoma. Thus, MYC maintains self-renewal exclusively in CSCs by selectively binding to the promoter and activating the HIF2a stemness pathway. Identification of this stemness pathway as a unique CSC determinant may have significant therapeutic implications.

We postulate that similar to bacteria, adult stem cells may also exhibit altruistic defense mechanism to protect their niche. Here, we provide preliminary data on the altruistic stem cell (ASC) based defense against a mouse corona virus; MHV-1 infection. In a mouse model of mesenchymal stem cell (MSC) mediated M. tuberculosis (Mtb) dormancy, MHV-1 infection in the lung exhibited 20 fold lower viral loads than the healthy control mice, suggesting the potential enhancement of an anti-MHV-1 defense by Mtb. This defense involved the in vivo expansion and reprogramming of CD271+ MSCs in the lung to ASC phenotype characterized by activation of genes involved in the HIF-2alpha stemness pathway. The conditioned media of the ASCs exhibited direct anti-viral activity in an in vitro model of MHV-1 induced toxicity to type II alveolar epithelial cells. MHV-1 infected Mtb harboring group versus MHV-1 alone groupexhibited an 8-fold (p<0.02; n=4) higher ASC reprogramming and 5-fold (p<0.001; n=3, student t test) higher anti-viral activity. However, ASCs facilitated intracellular replication and extracellular release of Mtb. Thus, our data suggest that MSCs exert an innate defense against MHV-1 by activating the ASC defense mechanism, which might be exploited by dormant Mtb to undergo reactivation. Hence, our findings may provide a novel anti-viral defense mechanism against novel corona virus SARS-Cov2, which could be further utilized to develop vaccine

Human bone marrow derived mesenchymal stem cells (BM-MSCs) resides in their niches in close proximity to hematopoietic stem cells (HSCs). These naïve MSCs have tremendous potential in regenerative therapeutics, and may also be exploited by cancer and infectious disease agents. Hence, it is important to study the physiological and pathological roles of naïve MSC. However, our knowledge of naïve MSCs is limited by lack of appropriate isolation and in vitro culture methods. Established culture methods use serum rich media, and serial passaging for retrospective isolation of MSCs. These primed MSCs may not reflect the true physiological and pathological roles of naive MSCs (Figure 1). Therefore, there is a strong need for direct isolation and in vitro culture of naïve MSCs to study their stemness (self-renewal and undifferentiated state) and developmental ontogeny. We have taken a niche-based approach on stemness to better maintain naïve MSCs in vitro. In this approach, stemness is broadly divided as niche dependent (extrinsic), niche independent (intrinsic) and niche modulatory (altruistic or competitive). Using this approach, we were able to maintain naïve CD271+/CD133+ BM-MSCs for 2 weeks. Furthermore, this in vitro culture system helped us to identify naïve MSCs as a protective niche site for Mycobacterium tuberculosis, the causative organism of pulmonary tuberculosis. In this review, we discuss the in vitro culture of primed vs. naïve human BM derived MSCs with a special focus on how a stemness based approach could facilitate the study of naïve BM-MSCs.

We postulate that similar to bacteria, adult stem cells may also exhibit an altruistic defense mechanism to protect their niche against external threat. Here, we report mesenchymal stem cell (MSC) based altruistic defense against a mouse model of coronavirus, murine hepatitis virus-1 (MHV-1) infection of lung. MHV-1 infection led to reprogramming of CD271+MSCs in the lung to an “enhanced stemness” phenotype that exhibits altruistic behavior as per our previous work in human embryonic stem cells. The reprogrammed MSCs exhibited transient expansion for two weeks followed by apoptosis, and expression of stemness genes The conditioned media of the reprogrammed MSCs exhibited direct anti-viral activity in an in vitro model of MHV-1 induced toxicity to type II alveolar epithelial cells by increasing their survival/proliferation and decreasing viral load. Thus, the reprogrammed MSCs can be identified as altruistic stem cells (ASCs) which exert a unique altruistic defense against MHV-1. In a mouse model of MSC mediated Mycobacterium tuberculosis ( Mtb ) dormancy, MHV-1 infection in the lung exhibited 20-fold lower viral loads than the Mtb -free control mice on the third week of viral infection, and also exhibited 6-fold increase of ASCs, thereby enhancing the altruistic defense. Notably, these ASCs exhibited intracellular replication of Mtb , and their extracellular release. Animals showed TB reactivation suggesting that d Mtb may exploit ASCs for disease reactivation..

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Stem cell may exhibit altruistic behavior that may benefit cancer cells. We recently demonstrated altruistic phenotype in human embryonic stem cells (Das B et al. Stem Cells 2012). The phenotype exhibited reversible induction of high HIF-2alpha and low p53 expression, associated with high glutathione secretion. We speculated that cancer stem cell might induce a similar altruistic phenotype in human bone marrow (BM) derived stem cells (hematopoietic, mesenchymal and endothelial cells). The altruistic reprogrammed BM cells may then facilitate tumor growth, as well as resist the toxicity of oxidative-stress inducing anti-cancer agents. To investigate this possibility, we have obtained conditioned media (CM) from migratory side-population (SPm) and non-SP cells of a diverse panel of tumors including epithelial tumors. The SPm cells exhibit very high tumorigenic capacity (Das B et al, Stem Cell, 2008). The CM was added to in vitro bone marrow (BM) derived CD133+ cells that contain hematopoietic, endothelial, and mesenchymal stem cells. We found that SPm derived CM (henceforth known as SPm-CM) treatment increased the self-renewal capacity of CD271+/CD45- BM-MSCs. Importantly, the reprogrammed CD271+ BM-MSCs (henceforth known as R-MSCs) phenotype exhibited enhanced stemness reprogramming, a cytoprotective mechanism associated with stem cell altruism (Das B et al. Stem Cells, 2012). In contrast, the treatment with CM obtained from non-SP cells did not exhibit R-MSCs. We found that VEGF/VEGR1 autocrine signaling may be involved in R-MSCs mechanism. Importantly, the R-MSCs derived CM reprogrammed non-CSCs to CSCs, and reduced the toxicity of chemotherapy on non-SP cells. In vivo, R-MSCs derived CM, when injected to mice, exhibited mobilization of CD271+ BM-MSCs to circulation. The circulatory CD271+ BM-MSCs exhibited distinct phenotype of R-MSCs including high expression of HIF-2alpha, and VEGFR1. Finally, in human cancer patients, we identified R-MSC phenotype in the peripheral circulation. These studies suggest that cancer stem cells may exploit stem cell altruism to reprogram BM-MSCs for their own benefit. The reprogrammed BM-MSCs gene expression may have the potential as a diagnostic marker for CSC-induced stem cell altruism. Citation Format: Joyeeta Talukdar, Rashmi Bhuyan, Jaishree Garhyan, Bidisha Pal, Sora Sandhya, Sukanya Gayan, Anupam Sarma, Reza Bayat-Mokhtari, Hong Li, Jyotirmoy Phukan, Wael Tasabehji, Seema Bhuyan, Amal Ch Kataki, Rika Tsuchida, Herman Yeger, Debabrata Baishya, Bikul Das. Migratory cancer side population cells induces stem cell altruism in bone marrow mesenchymal stem cells to resist therapy, and enhance tumorigenic potential of non-tumorigenic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 920.

Natural vaccination against pathogens are known to be achieved by herd-immunity i.e. infected human host provide immunity to the community by spreading the pathogen. Whether, infected human hosts transmit vesicle packed aerosols of pathogen’s antigen for natural vaccination of the community has not yet been considered. We have explored a traditional healing method of aerosol-inoculation against small pox and tuberculosis in the Sualkuchi-Hajo cultural complex of Kamarupa, an ancient Indian region known for tantra-based healing and spirituality. In the aerosol-inoculation method against TB, selected persons with TB (later identified as smear negative TB subject) are encouraged to spread good nigudah in the community by Kirtan chanting; the good Nigudah are thought to be present within bad-nigudah or invisible krimis (tiny flesh eating living being mentioned in ancient India’s medicinal text Caraka Samhita and Atharva Veda). A 15-years of contact TB investigation study, as well as laboratory study of aerosol obtained from smear negative PTB (SN-PTB) subjects led to the identification of good Nigudah as extracellular vesicles (EVs) filled with Mtb-antigen ESAT-6. We then developed a mouse model of aerosol-inoculation using SN-PTB subject derived aerosol EVs, and identified Mtb infected mesenchymal stem cells (MSCs) of the lung as the putative source of the ESAT-6+ EVs. These Mtb infected MSCs reprogram to altruistic stem cell (ASC) phenotype, which then secrete ESAT-6+ EVs to the aerosols; healthy mice receiving the aerosol develop Mtb specific herd immunity. These results expedite our ongoing work on the innate defense mechanism of ASCs against pathogen, and provide a novel mechanism of natural vaccination, where the host extracts appropriate antigens from a pathogen, and then spread it in the community via aerosols.

The mechanism of tumor hypoxia induced reprogramming of cancer cells are not understood well. Hypoxia might activate evolutionary preserved cellular defense mechanism that could contribute to tumor progression and metastasis. We recently described that during hypoxia/oxidative stress, human embryonic stem cells (hES) exhibit an altruistic defense mechanism, where a few cells reprogram to a highly undifferentiated state, and secrete glutathione to protect rest of the community of cells from oxidative stress induced DNA damage (Das B et al, Stem Cells, 2012). The altruistic stem cells phenotype exhibited high HIF-2alpha and low p53 activity. After a few weeks, the reprogrammed cells, although highly fit to survives, underwent spontaneous apoptosis/differentiation by re-activating the p53/MDM2 oscillation. Thus, the reprogrammed cells sacrificed its own fitness to enhance the fitness of the rest of the community, an altruistic behavior (Das B et al. Stem Cells, 2012). Here, we investigated the potential hijacking of the altruistic defense mechanism by oral cancer cells during exposure to hypoxia. We exposed the four oral cancer cell lines SCC-25, SCC-15, SCC-9 and SCC-5 to extreme hypoxia followed by re-oxygenation for 72 hours. We found that while majority of cancer cells underwent apoptosis, a few cancer cell lines survived, exhibited side-population (SP) phenotype, high level of HIF-2alpha, Nanog, Sox-2 and MYC. The SP cells exhibited migratory activity, as well as high tumorigenic and metastatic activity in NOD/SCID mice. ChIP assay indicated that HIF-2alhpa interact with MYC and NOTCH1. The conditioned media of SP cells exhibited high level of glutathione, and ability to protect non-SP cells from cisplatin-induced toxicity. These results indicate that HIF-2alpha and MYC may cooperate to reprogram a few oral cancer cells to altruistic stemness phenotype. The altruistic phenotype that exhibited cytoprotective activity against cisplatin mediated toxicity. Thus, similar to bacteria, where altruistic biofilm exhibit novel drug resistance mechanism, stem cell altruism may serve as a novel drug resistance mechanism in oral cancer. Citation Format: Rashmi Bhuyan, Hong Li, Sukanya Gayan, Bidisha Pal, Reza Bayat-Mokhtari, Jyotirmoy Phukan, Debabrata Baishya, Anupam Sarma, Joyeeta Talukdar, Manaf Muhammad Alkurdi, Wael Tasabehji, Seema Bhuyan, Gayatri Gogoi, Ista Pulu, Herman Yeger, Bikul Das. Oral cancer cells may hijack stem cell altruism to survive during extreme hypoxia, and exposure to chemotherapeutic drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 935.