Applying the Lean Startup Method to Structure Project-Based, Student-Driven Curricular Enhancements.

PROBLEM: Formal medical student engagement in curricular evaluation provides significant value through identification of opportunities for curricular change. Students provide diverse perspectives and have a unique vantage point, which allows them to see aspects of the curriculum that educators and administrators might not recognize. Current descriptions of student engagement are focused largely on collection, analysis, and presentation of summative feedback in the pre-clerkship curriculum. However, medical students could potentially contribute to curricular improvement in ways extending beyond post hoc curricular evaluation. Student teams focused on identification of specific needs and project-based implementation of solutions represent one means of doing so but require a structured, organizing method in order to succeed. INTERVENTION: We describe a novel, project-based, student-driven medical education initiative, the Special Projects Team, which is focused on identifying opportunities for forward-looking curricular enhancements beyond single courses or rotations. We adapted and implemented the lean startup method, a model for project management, in order to address the need for organization and accountability in the Special Projects Team. Members of the Special Projects Team were recruited from the first- and second-year medical school classes in the 2018-2020 academic years and provided with training on the lean startup method. Team members selected and pursued projects according to the principles of lean startup method, reporting their progress to the chair of the Special Projects Team and other team members at monthly meetings with pre-defined structure. CONTEXT: The Special Projects Team is part of the local Student Curricular Board at the Chicago campus of the University of Illinois College of Medicine. The Student Curricular Board is responsible for conducting curricular evaluation and improvement, operating under the local medical student council with financial support from the Office of Curricular Affairs. Direct supervision of the Special Projects Team is provided by a student chair, the executive board of the Student Curricular Board, and the curricular dean. IMPACT: The projects initiated as part of the Special Projects Team covered a broad range of themes, including curricular evaluation, technology, and student experiences. Lean startup method contributed to sustained project success and frequent reassessment across the two years of our experience, with aggregate project success or continuation rate of 68.4% (13/19 projects). We further demonstrate how lean startup method increased productivity while providing structure and accountability for a student-led medical education team. LESSONS LEARNED: Lean startup method can be used to structure student-driven, project-based curricular enhancements. This approach is broadly applicable to other medical schools with implementation requiring only a motivated student team, faculty advisor, and basic knowledge of the lean startup method.

Elucidating Extracellular Matrix and Stiffness Control of Primary Human Hepatocyte Phenotype Via Cell Microarrays.

How the liver's extracellular matrix (ECM) protein composition and stiffness cooperatively regulate primary human hepatocyte (PHH) phenotype is unelucidated. Here, we utilize protein microarrays and high content imaging with single-cell resolution to assess PHH attachment/functions on 10 major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of 1 kPa or 25 kPa stiffness. Albumin, cytochrome-P450 3A4 (CYP3A4), and hepatocyte nuclear factor alpha (HNF4α) positively correlate with each other and cell density on both stiffnesses. The 25 kPa stiffness supports higher average albumin and HNF4α expression after 14 days, while ECM protein composition significantly modulates PHH functions across both stiffnesses. Unlike previous rodent data, PHH functions are highest only when collagen-IV or fibronectin are mixed with specific proteins, whereas non-collagenous proteins without mixed collagens downregulate functions. Combination of collagen-IV and hyaluronic acid retains high CYP3A4 on 1 kPa, whereas collagens-IV and -V better retain HNF4α on 25 kPa over 14 days. Adapting ECM conditions to 96-well plates containing conjugated hydrogels reveals novel regulation of other functions (urea, CYP1A2/2A6/2C9) and drug-mediated CYP induction by the ECM protein composition/stiffness. This high-throughput pipeline can be adapted to elucidate ECM's role in liver diseases and facilitate optimization of engineered tissues.

Simultaneous robotic kidney transplantation and bariatric surgery for morbidly obese patients with end-stage renal failure.

Patients with obesity have limited access to kidney transplantation, mainly due to an increased incidence of surgical complications, which could be reduced with selective use of robotic-assisted surgery. This prospective randomized controlled trial compares the safety and efficacy of combining robotic sleeve gastrectomy and robotic-assisted kidney transplant to robotic kidney transplant alone in candidates with class II or III obesity. Twenty candidates were recruited, 11 were randomized to the robotic sleeve gastrectomy and robotic-assisted kidney transplant group and 9 to the robotic kidney transplant group. At 12-month follow-up, change in body mass index was -8.76 ± 1.82 in the robotic sleeve gastrectomy and robotic-assisted kidney transplant group compared to 1.70 ± 2.30 in the robotic kidney transplant group (P = .0041). Estimated glomerular filtration rate, serum creatinine, readmission rates, and graft failure rates up to 12 months were not different between the two groups. Length of surgery was longer in the robotic sleeve gastrectomy and robotic-assisted kidney transplant group (405 minutes vs. 269 minutes, p = .00304) without increase in estimated blood loss (120 ml vs. 117 ml, p = .908) or incidence of surgical complications. Combined robotic-assisted kidney transplant and sleeve gastrectomy is safe and effective compared to robotic-assisted kidney transplant alone.

Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling.

The progenitor cells of the developing liver can differentiate toward both hepatocyte and biliary cell fates. In addition to the established roles of TGFß and Notch signaling in this fate specification process, there is increasing evidence that liver progenitors are sensitive to mechanical cues. Here, we utilized microarrayed patterns to provide a controlled biochemical and biomechanical microenvironment for mouse liver progenitor cell differentiation. In these defined circular geometries, we observed biliary differentiation at the periphery and hepatocytic differentiation in the center. Parallel measurements obtained by traction force microscopy showed substantial stresses at the periphery, coincident with maximal biliary differentiation. We investigated the impact of downstream signaling, showing that peripheral biliary differentiation is dependent not only on Notch and TGFß but also E-cadherin, myosin-mediated cell contractility, and ERK. We have therefore identified distinct combinations of microenvironmental cues which guide fate specification of mouse liver progenitors toward both hepatocyte and biliary fates.

Cellular fate decisions in the developing female anteroventral periventricular nucleus are regulated by canonical Notch signaling.

The hypothalamic anteroventral periventricular nucleus (AVPV) is the major regulator of reproductive function within the hypothalamic-pituitary-gonadal (HPG) axis. Despite an understanding of the function of neuronal subtypes within the AVPV, little is known about the molecular mechanisms regulating their development. Previous work from our laboratory has demonstrated that Notch signaling is required in progenitor cell maintenance and formation of kisspeptin neurons of the arcuate nucleus (ARC) while simultaneously restraining POMC neuron number. Based on these findings, we hypothesized that the Notch signaling pathway may act similarly in the AVPV by promoting development of kisspeptin neurons at the expense of other neuronal subtypes. To address this hypothesis, we utilized a genetic mouse model with a conditional loss of Rbpj in Nkx2.1 expressing cells (Rbpj cKO). We noted an increase in cellular proliferation, as marked by Ki-67, in the hypothalamic ventricular zone (HVZ) in Rbpj cKO mice at E13.5. This corresponded to an increase in general neurogenesis and more TH-positive neurons. Additionally, an increase in OLIG2-positive early oligodendrocytic precursor cells was observed at postnatal day 0 in Rbpj cKO mice. By 5 weeks of age in Rbpj cKO mice, TH-positive cells were readily detected in the AVPV but few kisspeptin neurons were present. To elucidate the direct effects of Notch signaling on neuron and glia differentiation, an in vitro primary hypothalamic neurosphere assay was employed. We demonstrated that treatment with the chemical Notch inhibitor DAPT increased mKi67 and Olig2 mRNA expression while decreasing astroglial Gfap expression, suggesting Notch signaling regulates both proliferation and early glial fate decisions. A modest increase in expression of TH in both the cell soma and neurite extensions was observed after extended culture, suggesting that inhibition of Notch signaling alone is enough to bias progenitors towards a dopaminergic fate. Together, these data suggest that Notch signaling restricts early cellular proliferation and differentiation of neurons and oligodendrocytes both in vivo and in vitro and acts as a fate selector of kisspeptin neurons.

Substrate stiffness and VE-cadherin mechano-transduction coordinate to regulate endothelial monolayer integrity.

The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial barrier function. In addition to rigidity sensing through integrin adhesions, mechanical perturbations such as changes in fluid shear stress can also activate force transduction signals at intercellular junctions. This study investigated how extracellular matrix rigidity and intercellular force transduction, activated by vascular endothelial cadherin, coordinate to regulate the integrity of endothelial monolayers. Studies used complementary mechanical measurements of endothelial monolayers grown on patterned substrates of variable stiffness. Specifically perturbing VE-cadherin receptors activated intercellular force transduction signals that increased integrin-dependent cell contractility and disrupted cell-cell and cell-matrix adhesions. Further investigations of the impact of substrate rigidity on force transduction signaling demonstrated how cells integrate extracellular mechanics cues and intercellular force transduction signals, to regulate endothelial integrity and global tissue mechanics. VE-cadherin specific signaling increased focal adhesion remodeling and cell contractility, while sustaining the overall mechanical equilibrium at the mesoscale. Conversely, increased substrate rigidity exacerbates the disruptive effects of intercellular force transduction signals, by increasing heterogeneity in monolayer stress distributions. The results provide new insights into how substrate stiffness and intercellular force transduction coordinate to regulate endothelial monolayer integrity.

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell-substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young's modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Mapping lung tumor cell drug responses as a function of matrix context and genotype using cell microarrays.

Carcinoma progression is influenced by interactions between epithelial tumor cells and components of their microenvironment. In particular, cell-extracellular matrix (ECM) interactions are known to drive tumor growth, metastatic potential, and sensitivity or resistance to therapy. Yet the intrinsic complexity of ECM composition within the tumor microenvironment remains a barrier to comprehensive investigation of these interactions. We present here a high-throughput cell microarray-based approach to study the impact of defined combinations of ECM proteins on tumor cell drug responses. Using this approach, we quantitatively evaluated the effects of 55 different ECM environments representing all single and two-factor combinations of 10 ECM proteins on the responses of lung adenocarcinoma cells to a selection of cancer-relevant small molecule drugs. This drug panel consisted of an alkylating agent and five receptor tyrosine kinase inhibitors. We further determined that expression of the neuroendocrine transcription factor ASCL1, which has been previously associated with poor patient outcome when co-expressed with the RET oncogene, altered cell responses to drugs and modulated cleavage of the pro-apoptotic protein caspase-3 depending on ECM context. Our results suggest that co-expression of specific ECM proteins with known genetic drivers in lung adenocarcinoma may impact therapeutic efficacy. Furthermore, this approach could be utilized to define the molecular mechanisms by which cell-matrix interactions drive drug resistance through integration with clinical cell samples and genomics data.

Substrate stiffness and matrix composition coordinately control the differentiation of liver progenitor cells.

Recent approaches have utilized microfabricated platforms to examine combinations of microenvironmental signals that regulate stem and progenitor cell differentiation. However, the majority of these efforts have focused on the biochemical properties of extracellular matrix (ECM) or soluble factors without simultaneously exploring the biomechanical effects of cell-substrate interactions. To address this need, we combined a high-throughput approach for the analysis of combinatorial ECM cues with substrates of modular stiffness and traction force microscopy. This integrated approach enabled the characterization of cell-generated traction stress and phenotypic expression in response to ECM cues. We investigated the impact of substrate stiffness and ECM composition on the differentiation of bipotential mouse embryonic liver (BMEL) progenitor cells. We observed that hepatocyte differentiation was primarily regulated by ECM composition, and cholangiocyte differentiation was cooperatively influenced by ECM proteins and stiffness properties. In particular, stiffness-mediated cholangiocyte differentiation was observed for cells cultured on fibronectin, while collagen IV promoted differentiation independent of substrate stiffness. We demonstrated the influence of cell contractility and traction stress in early cholangiocyte specification and further uncovered the roles of ERK and ROCK in this differentiation process. Overall, these findings illustrate the involvement of biomechanical signals in liver progenitor differentiation. Further, this approach could enable investigations for a broad range of cell types and ECM proteins, providing an integrated platform for evaluating the combinatorial effects of biochemical and biophysical signals in cell differentiation.

Combinatorial microenvironmental regulation of liver progenitor differentiation by Notch ligands, TGFß, and extracellular matrix.

The bipotential differentiation of liver progenitor cells underlies liver development and bile duct formation as well as liver regeneration and disease. TGFß and Notch signaling are known to play important roles in the liver progenitor specification process and tissue morphogenesis. However, the complexity of these signaling pathways and their currently undefined interactions with other microenvironmental factors, including extracellular matrix (ECM), remain barriers to complete mechanistic understanding. Utilizing a series of strategies, including co-cultures and cellular microarrays, we identified distinct contributions of different Notch ligands and ECM proteins in the fate decisions of bipotential mouse embryonic liver (BMEL) progenitor cells. In particular, we demonstrated a cooperative influence of Jagged-1 and TGFß1 on cholangiocytic differentiation. We established ECM-specific effects using cellular microarrays consisting of 32 distinct combinations of collagen I, collagen III, collagen IV, fibronectin, and laminin. In addition, we demonstrated that exogenous Jagged-1, Delta-like 1, and Delta-like 4 within the cellular microarray format was sufficient for enhancing cholangiocytic differentiation. Further, by combining Notch ligand microarrays with shRNA-based knockdown of Notch ligands, we systematically examined the effects of both cell-extrinsic and cell-intrinsic ligand. Our results highlight the importance of divergent Notch ligand function and combinatorial microenvironmental regulation in liver progenitor fate specification.