Differentiation of monocytes and polarized M1/M2 macrophages from human induced pluripotent stem cells.

Here, we present a protocol to differentiate induced pluripotent stem cell (iPSC) into adherent hematopoietic progenitors that release floating CD14+ CD45+ monocytes into the culture medium. We describe steps for iPSC expansion, embryoid body (EB) formation, suspension culture, plating EBs, and recurring harvests of monocytes, a.k.a. "monocyte factory." We then describe detailed procedures for freezing/thawing of monocytes and differentiation into polarized M1 and M2 macrophages. This protocol provides foundation to study iPSC monocytes and their progenies such as macrophages, microglial, and dendritic cells. For complete details on the use and execution of this protocol, please refer to Karlson et al.1 and Panicker et al.2.

Retroviral infection of human neurospheres and use of stem Cell EVs to repair cellular damage.

HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection.

Engineered induced-pluripotent stem cell derived monocyte extracellular vesicles alter inflammation in HIV humanized mice.

Aim: A peripheral inflammatory response can drive neuroinflammation in a number of infections including human immunodeficiency virus (HIV). Monocyte/macrophage (M/Mφ) activation is a hallmark of acute HIV infection and a source of chronic inflammation in a subset of HIV-infected individuals. We sought to decrease peripheral inflammation and M/Mφ transmigration after HIV infection by engineering extracellular vesicles (EV) to antagonize a microRNA (miR) associated with inflammation. We hypothesized that induced pluripotent stem cell (iPSC)-derived monocyte EVs (mEVs), engineered to contain an antagomir to miR-155 (αmiR mEV) would target monocyte inflammation and influence neuroinflammation in an HIV-infected humanized mice. Methods: mEVs were characterized by tetraspanins, nanoparticle tracking analysis, electron microscopy, and their preferential entry into circulating monocytes as well as testing for endogenous selected miRNAs. HIV-infected humanized mice were treated with control or antagomir155 mEVs. Plasma viral load was measured plus activation markers on lymphocytes and monocytes and the number of macrophages in the brain was quantified. Results: mEVs preferentially entered peripheral monocytes. HIV infection increased C-C chemokine receptor type 5 (CCR5) and major histocompatibility complex, class II, DR (HLA-DR) expression on T cells and monocytes. Treatments with mEVs did not decrease plasma HIV viral load; however, mEVs alone resulted in a decrease in %CCR5+ and %HLA-DR+ on T cells and an increase in %CCR5+ monocytes. αmiR mEVs decreased %CCR5 on M/Mφ. The mEV-treated HIV-infected mice did not show an increase in macrophage transmigration to the brain. Conclusion: mEVs alone caused an unexpected decrease in lymphocyte activation and increase in monocyte %CCR5; however, this did not translate to an increase in macrophage transmigration to the brain.