Kelly A. Purpura

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.

Don't Look: Growing Clonal Versus Nonclonal Neural Stem Cell Colonies

Recent reports have challenged the clonality of the neurosphere assay in assessing neural stem cell (NSC) numbers quantitatively. We tested the clonality of the neurosphere assay by culturing mixtures of differently labeled neural cells, watching single neural cells proliferate using video microscopy, and encapsulating single NSCs and their progeny. The neurosphere assay gave rise to clonal colonies when using primary cells plated at 10 cells/μl or less; however, when using passaged NSCs, the spheres were clonal only if plated at 1 cell/μl. Most important, moving the plates during the growth phase (to look at cultures microscopically) greatly increased the incidence of nonclonal colonies. To ensure clonal sphere formation and investigate nonautonomous effects on clonal sphere formation frequencies, single NSCs were encapsulated in agarose and proliferated as clonal free‐floating spheres. We demonstrate that clonal neurospheres can be grown by avoiding movement‐induced aggregation, by single‐cell tracking, and by encapsulation of single cells.

Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent

Osteogenic cells are an integral part of the dynamic tissue‐remodeling process in bone and are potential tools for tissue engineering and cell‐based therapies. We examined the role of glucocorticoids and cell density in the expansion of primary rat calvaria cell populations and osteoprogenitor subpopulations in adherent cell culture. Osteoprogenitor response to dexamethasone (dex, a synthetic glucocorticoid known to stimulate bone formation in vitro) supplementation and long‐term osteoprogenitor cell proliferation and differentiation were quantified using functional (colony forming unit‐osteoblast [CFU‐O]) and phenotypic analyses. Although osteoprogenitor self‐renewal occurred at both standard and high initiating cell densities, progenitor cell expansion (measured by changes in CFU‐O number relative to input) was sustained and dramatically increased at high initiating cell densities (30‐fold CFU‐O expansion for standard‐density cultures compared with a greater than 10,000‐fold CFU‐O expansion in high‐density cultures). Cell density was also found to impact upon the potential of dex to recruit additional progenitors towards bone development. These multifaceted effects appeared to be independent of cell proliferation rates or population phenotypic expression. Together, our results emphasize a roll for cell‐cell interactions and/or community effects in the control and maintenance of progenitor cells during in vitro culture.

The use of vascular endothelial growth factor functionalized agarose to guide pluripotent stem cell aggregates toward blood progenitor cells

The developmental potential of pluripotent stem cells is influenced by their local cellular microenvironment. To better understand the role of vascular endothelial growth factor (VEGFA) in the embryonic cellular microenvironment, we synthesized an artificial stem cell niche wherein VEGFA was immobilized in an agarose hydrogel. Agarose was first modified with coumarin-protected thiols. Upon exposure to ultra-violet excitation, the coumarin groups were cleaved leaving reactive thiols to couple with maleimide-activated VEGFA. Mouse embryonic stem cells (ESC) aggregates were encapsulated in VEGFA immobilized agarose and cultured for 7 days as free-floating aggregates under serum-free conditions. Encapsulated aggregates were assessed for their capacity to give rise to blood progenitor cells. In the presence of bone morphogenetic protein-4 (BMP-4), cells exposed to immobilized VEGFA upregulated mesodermal markers, brachyury and VEGF receptor 2 (T+VEGFR2+) by day 4, and expressed CD34 and CD41 (CD34+CD41+) on day 7. It was found that immobilized VEGFA treatment was more efficient at inducing blood progenitors (including colony forming cells) on a per molecule basis than soluble VEGFA. This work demonstrates the use of functionalized hydrogels to guide encapsulated ESCs toward blood progenitor cells and introduces a tool capable of recapitulating aspects of the embryonic microenvironment.

Soluble Flt-1 regulates Flk-1 activation to control hematopoietic and endothelial development in an oxygen-responsive manner.

Vascular endothelial growth factor (VEGF) and the vascular endothelial growth factor receptors (VEGFRs) regulate the development of hemogenic mesoderm. Oxygen concentration‐mediated activation of hypoxia‐inducible factor targets such as VEGF may serve as the molecular link between the microenvironment and mesoderm‐derived blood and endothelial cell specification. We used controlled‐oxygen microenvironments to manipulate the generation of hemogenic mesoderm and its derivatives from embryonic stem cells. Our studies revealed a novel role for soluble VEGFR1 (sFlt‐1) in modulating hemogenic mesoderm fate between hematopoietic and endothelial cells. Parallel measurements of VEGF and VEGFRs demonstrated that sFlt‐1 regulates VEGFR2 (Flk‐1) activation in both a developmental‐stage‐dependent and oxygen‐dependent manner. Early transient Flk‐1 signaling occurred in hypoxia because of low levels of sFlt‐1 and high levels of VEGF, yielding VEGF‐dependent generation of hemogenic mesoderm. Sustained (or delayed) Flk‐1 activation preferentially yielded hemogenic mesoderm‐derived endothelial cells. In contrast, delayed (sFlt‐1‐mediated) inhibition of Flk‐1 signaling resulted in hemogenic mesoderm‐derived blood progenitor cells. Ex vivo analyses of primary mouse embryo‐derived cells and analysis of transgenic mice secreting a Flt‐1‐Fc fusion protein (Fc, the region of an antibody which is constant and binds to receptors) support a hypothesis whereby microenvironmentally regulated blood and endothelial tissue specification is enabled by the temporally variant control of the levels of Flk‐1 activation.

Systematic engineering of 3D pluripotent stem cell niches to guide blood development

Pluripotent stem cells (PSC) provide insight into development and may underpin new cell therapies, yet controlling PSC differentiation to generate functional cells remains a significant challenge. In this study we explored the concept that mimicking the local in vivo microenvironment during mesoderm specification could promote the emergence of hematopoietic progenitor cells from embryonic stem cells (ESCs). First, we assessed the expression of early phenotypic markers of mesoderm differentiation (E-cadherin, brachyury (T-GFP), PDGFRα, and Flk1: +/-ETPF) to reveal that E-T+P+F+ cells have the highest capacity for hematopoiesis. Second, we determined how initial aggregate size influences the emergence of mesodermal phenotypes (E-T+P+F+, E-T-P+/-F+, and E-T-P+F-) and discovered that colony forming cell (CFC) output was maximal with ~100 cells per PSC aggregate. Finally, we introduced these 100-cell PSC aggregates into a low oxygen environment (5%; to upregulate endogenous VEGF secretion) and delivered two potent blood-inductive molecules, BMP4 and TPO (bone morphogenetic protein-4 and thrombopoietin), locally from microparticles to obtain a more robust differentiation response than soluble delivery methods alone. Approximately 1.7-fold more CFCs were generated with localized delivery in comparison to exogenous delivery, while combined growth factor use was reduced ~14.2-fold. By systematically engineering the complex and dynamic environmental signals associated with the in vivo blood developmental niche we demonstrate a significant role for inductive endogenous signaling and introduce a tunable platform for enhancing PSC differentiation efficiency to specific lineages.

Analysis of the temporal and concentration-dependent effects of BMP-4, VEGF, and TPO on development of embryonic stem cell–derived mesoderm and blood progenitors in a defined, serum-free media

OBJECTIVE To develop a robust serum-free (SF) system for generation of hemogenic mesoderm and blood progenitors from pluripotent cells. MATERIALS AND METHODS Embryonic stem cells (ESCs) maintained in N2B27 supplemented with leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-4 were induced to differentiate into Brachyury/T-expressing cells (measured using a green fluorescent protein reporter) and myeloid-erythroid colony-forming cells (ME-CFCs), by removing LIF, changing the base media formulation, and via the time- and concentration-dependent addition of other factors. RESULTS Presence of 10 ng/mL BMP-4 permitted the emergence of cells expressing T and the vascular endothelial growth factor receptor (VEGFR)-2, however, <5% of the cells were double-positive on day 4. Adjusting the SF media formulation allowed only 5 ng/mL BMP-4 to yield 24% +/- 4% Brachyury-green fluorescent protein VEGFR-2(+) cells by day 4. These cells could develop into ME-CFC, producing 4.4 +/- 0.8 CFC per 1000 cells at day 8. We also examined the timing and concentration sensitivity of BMP-4, VEGF, and thrombopoietin (TPO) during differentiation. BMP-4 with 50 ng/mL TPO generated 232 +/- 48 CFC per 5 x 10(4) cells, similar to the serum-control, and this response could be enhanced to 292 +/- 42 CFC per 5 x 10(4) cells by early (between day 0-5), but not late (after day 5) VEGF treatment. CONCLUSION Moving to SF systems facilitates directed differentiation by eliminating confounding signals. This article describes modifications to the N2B27 media that amplify mesoderm induction and extends earlier work defining blood progenitor cell induction from ESC with BMP-4, VEGF, and TPO.

Fluorescence Activated Cell Sorting Reveals Heterogeneous and Cell Non-Autonomous Osteoprogenitor Differentiation in Fetal Rat Calvaria Cell Populations

Identification of osteoblast progenitors, with defined developmental capacity, would facilitate studies on a variety of parameters of bone development. We used expression of alkaline phosphatase (ALP) and the parathyroid hormone/parathyroid hormone‐related protein receptor (PTH1R) as osteoblast markers in dual‐color fluorescence activated cell sorting (FACS) to fractionate rat calvaria (RC) cells into ALP−PTH1R−, ALP+PTH1R−, ALP−PTH1R+, and ALP+PTH1R+ populations. These fractionated populations were seeded clonally (n = 96) or over a range of cell densities (∼150–8,500 cell/cm2; n = 3). Our results indicate that colony forming unit‐osteoblast (CFU‐O)/bone nodule‐forming cells are found in all fractions, but the frequency of CFU‐O and total mineralized area is different across fractions. Analysis of these differences suggests that ALP−PTH1R−, ALP−PTH1R+, ALP+PTH1R−, and ALP+PTH1R+ cell populations are separated in order of increasing bone formation capacity. Dexamethasone (dex) differentially increased the CFU‐O number in the four fractions, with the largest stimulation in the ALP− cell populations. However, there was no significant difference in the number or size distribution of CFU‐F (fibroblast) colonies that formed in vehicle versus dex. Finally, both cell autonomous and cell non‐autonomous (i.e., inhibitory/stimulatory effects of cell neighbors) differentiation of osteoprogenitors was seen. Only the ALP−PTH1R− population was capable of forming nodules at the clonal level, at approximately 3‐ or 12‐times the predicted frequency of unfractionated populations in dex or vehicle, respectively. These data suggest that osteoprogenitors can be significantly enriched by fractionation of RC populations, that assay conditions modify the osteoprogenitor frequencies observed and that fractionation of osteogenic populations is useful for interrogation of their developmental status and osteogenic capacity. J. Cell. Biochem. 90: 109–120, 2003.