Raheem Peerani

TAZ controls Smad nucleocytoplasmic shuttling and regulates human embryonic stem-cell self-renewal

Transforming growth fazctor-β (TGFβ) family members regulate many developmental and pathological events through Smad transcriptional modulators. How nuclear accumulation of Smad is coupled to the transcriptional machinery is poorly understood. Here we demonstrate that in response to TGFβ stimulation the transcriptional regulator TAZ binds heteromeric Smad2/3–4 complexes and is recruited to TGFβ response elements. In human embryonic stem cells TAZ is required to maintain self-renewal markers and loss of TAZ leads to inhibition of TGFβ signalling and differentiation into a neuroectoderm lineage. In the absence of TAZ, Smad2/3–4 complexes fail to accumulate in the nucleus and activate transcription. Furthermore, TAZ, which itself engages in shuttling, dominantly controls Smad nucleocytoplasmic localization and can be retained in the nucleus by transcriptional co-factors such as ARC105, a component of the Mediator complex. TAZ thus defines a hierarchical system regulating Smad nuclear accumulation and coupling to the transcriptional machinery.

Niche-mediated control of human embryonic stem cell self-renewal and differentiation

Complexity in the spatial organization of human embryonic stem cell (hESC) cultures creates heterogeneous microenvironments (niches) that influence hESC fate. This study demonstrates that the rate and trajectory of hESC differentiation can be controlled by engineering hESC niche properties. Niche size and composition regulate the balance between differentiation‐inducing and ‐inhibiting factors. Mechanistically, a niche size‐dependent spatial gradient of Smad1 signaling is generated as a result of antagonistic interactions between hESCs and hESC‐derived extra‐embryonic endoderm (ExE). These interactions are mediated by the localized secretion of bone morphogenetic protein‐2 (BMP2) by ExE and its antagonist, growth differentiation factor‐3 (GDF3) by hESCs. Micropatterning of hESCs treated with small interfering (si) RNA against GDF3, BMP2 and Smad1, as well treatments with a Rho‐associated kinase (ROCK) inhibitor demonstrate that independent control of Smad1 activation can rescue the colony size‐dependent differentiation of hESCs. Our results illustrate, for the first time, a role for Smad1 in the integration of spatial information and in the niche‐size‐dependent control of hESC self‐renewal and differentiation.

Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])‐induced differentiation, a common approach to mimic some of the spatial and temporal aspects of in vivo development, are affected by three factors: input hESC composition, input hESC colony size, and EB size. Using a microcontact printing approach, size‐specified hESC colonies were formed by plating single‐cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently, size‐controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP‐hESC populations revealed that the ratio of Gata6 (endoderm‐associated marker) to Pax6 (neural‐associated marker) expression increased with decreasing colony size. Moreover, upon forming EBs from these MP‐hESCs, we observed that differentiation trajectories were affected by both colony and EB size‐influenced parameters. In MP‐EBs generated from endoderm‐biased (high Gata6/Pax6) input hESCs, higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely, neural‐biased (low Gata6/Pax6) input hESCs generated MP‐EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size, typical in most differentiation strategies, produces subsets of appropriate conditions for differentiation into specific cell types. Moreover, our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories.

Generation of human embryonic stem cell‐derived mesoderm and cardiac cells using size‐specified aggregates in an oxygen‐controlled bioreactor

The ability to generate human pluripotent stem cell‐derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and biopharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called “embryoid bodies” (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro‐printing strategy to generate large numbers of size‐specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size‐control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT‐PCR data for markers such as Brachyury, LIM domain homeobox gene Isl‐1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust generation of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493–507.

Enabling stem cell therapies through synthetic stem cell–niche engineering

Enabling stem cell-targeted therapies requires an understanding of how to create local microenvironments (niches) that stimulate endogenous stem cells or serve as a platform to receive and guide the integration of transplanted stem cells and their derivatives. In vivo, the stem cell niche is a complex and dynamic unit. Although components of the in vivo niche continue to be described for many stem cell systems, how these components interact to modulate stem cell fate is only beginning to be understood. Using the HSC niche as a model, we discuss here microscale engineering strategies capable of systematically examining and reconstructing individual niche components. Synthetic stem cell-niche engineering may form a new foundation for regenerative therapies.

High-throughput combinatorial cell co-culture using microfluidics

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate.

Micropatterning of human embryonic stem cells dissects the mesoderm and endoderm lineages.

Human pluripotent cells such as human embryonic stem cells (hESC) are a great potential source of cells for cell-based therapies; however, directing their differentiation into the desired cell types with high purity remains a challenge. The stem cell microenvironment plays a vital role in directing hESC fate and we have previously shown that manipulation of colony size in a serum- and cytokine-free environment controls self-renewal and differentiation toward the extraembryonic endoderm lineage. Here we show that, in the presence of bone morphogenetic protein 2 and activin A, control of colony size using a microcontact printing technology is able to direct hESC fate to either the mesoderm or the endoderm lineage. Large, 1200-mum-diameter colonies give rise to mesoderm, while small 200-mum colonies give rise to definitive endoderm. This study links, for the first time, cellular organization to pluripotent cell differentiation along the mesoderm and endoderm lineages.

Manipulation of Signaling Thresholds in ''Engineered Stem Cell Niches'' Identifies Design Criteria for Pluripotent Stem Cell Screens

In vivo, stem cell fate is regulated by local microenvironmental parameters. Governing parameters in this stem cell niche include soluble factors, extra-cellular matrix, and cell-cell interactions. The complexity of this in vivo niche limits analyses into how individual niche parameters regulate stem cell fate. Herein we use mouse embryonic stem cells (mESC) and micro-contact printing (µCP) to investigate how niche size controls endogenous signaling thresholds. µCP is used to restrict colony diameter, separation, and degree of clustering. We show, for the first time, spatial control over the activation of the Janus kinase/signal transducer and activator of transcription pathway (Jak-Stat). The functional consequences of this niche-size-dependent signaling control are confirmed by demonstrating that direct and indirect transcriptional targets of Stat3, including members of the Jak-Stat pathway and pluripotency-associated genes, are regulated by colony size. Modeling results and empirical observations demonstrate that colonies less than 100 µm in diameter are too small to maximize endogenous Stat3 activation and that colonies separated by more than 400 µm can be considered independent from each other. These results define parameter boundaries for the use of ESCs in screening studies, demonstrate the importance of context in stem cell responsiveness to exogenous cues, and suggest that niche size is an important parameter in stem cell fate control.

Sudden Death of a Young Man by Acute Hemorrhagic Leukoencephalitis

We report a case of acute hemorrhagic leukoencephalitis in an adult man with a prodrome of “feeling unwell” two days prior to this death. At autopsy, external examination revealed minor external injuries including contusions on the scalp and left thigh and abrasions on the forehead and right eyebrow. Gross examination of the brain after coronal sectioning revealed multiple petechial hemorrhages in the white matter in the cerebral hemispheres, corpus callosum, basal ganglia, brainstem, and cerebellum. Microscopic examination of these lesions revealed demyelination, hemorrhage, and necrosis with fibrin exudation in a perivenular distribution with radial extension into the white matter. The remainder of the autopsy was unremarkable. This case highlights the death of a young man by a rare fatal complication of a natural disease only identified by a singular set of gross and microscopic findings at autopsy in circumstances that would otherwise suggest a nonnatural death. The case demonstrates the importance of a thorough autopsy in settings where the clinical history, scene, and circumstances may be misleading or absent.