Adult Stem Cell Plasticity Research Articles

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

Core transcription regulatory circuitry orchestrates corneal epithelial homeostasis

Adult stem cell identity, plasticity, and homeostasis are precisely orchestrated by lineage-restricted epigenetic and transcriptional regulatory networks. Here, by integrating super-enhancer and chromatin accessibility landscapes, we delineate core transcription regulatory circuitries (CRCs) of limbal stem/progenitor cells (LSCs) and find that RUNX1 and SMAD3 are required for maintenance of corneal epithelial identity and homeostasis. RUNX1 or SMAD3 depletion inhibits PAX6 and induces LSCs to differentiate into epidermal-like epithelial cells. RUNX1, PAX6, and SMAD3 (RPS) interact with each other and synergistically establish a CRC to govern the lineage-specific cis-regulatory atlas. Moreover, RUNX1 shapes LSC chromatin architecture via modulating H3K27ac deposition. Disturbance of RPS cooperation results in cell identity switching and dysfunction of the corneal epithelium, which is strongly linked to various human corneal diseases. Our work highlights CRC TF cooperativity for establishment of stem cell identity and lineage commitment, and provides comprehensive regulatory principles for human stratified epithelial homeostasis and pathogenesis.

Exploring Notch Pathway to Elucidate Phenotypic Plasticity and Intra-tumor Heterogeneity in Gliomas

The phenotypic plasticity and self-renewal of adult neural (aNSCs) and glioblastoma stem cells (GSCs) are both known to be governed by active Notch pathway. During development, GSCs can establish differential hierarchy to produce heterogeneous groups of tumor cells belong to different grades, which makes the tumor ecosystem more complex. However, the molecular events regulating these entire processes are unknown hitherto. In this work, based on the mechanistic regulations of Notch pathway activities, a novel computational framework is introduced to inspect the intra-cellular reactions behind the development of normal and tumorigenic cells from aNSCs and GSCs, respectively. The developmental dynamics of aNSCs/GSCs are successfully simulated and molecular activities regulating the phenotypic plasticity and self-renewal processes in normal and tumor cells are identified. A novel scoring parameter “Activity Ratio” score is introduced to find out driver molecules responsible for the phenotypic plasticity and development of different grades of tumor. A new quantitative method is also developed to predict the future risk of Glioblastoma tumor of an individual with appropriate grade by using the transcriptomics profile of that individual as input. Also, a novel technique is introduced to screen and rank the potential drug-targets for suppressing the growth and differentiation of tumor cells.

Adult neural stem cell plasticity.

Adult neural stem cell plasticity.

Phenotype and multipotency of rabbit (Oryctolagus cuniculus) amniotic stem cells

BackgroundStem cells are capable of unlimited self-renewal and are able to remain undifferentiated for extended periods of time prior to their differentiation into specific cell lineages. Because of the issues (ethical and religious) involved in the use of embryonic stem cells and the limited plasticity of adult stem cells, an alternative cell source could be foetal stem cells derived from extra-embryonic tissue, which are highly proliferative, grow in vitro and possess interesting immunogenic characteristics. As a result, the amniotic membrane of several species has been studied as an important new source of stem cells.MethodsHere, we cultured and characterized mesenchymal progenitor cells derived from the rabbit amniotic membrane, and investigated their differentiation potential. In total, amniotic membranes were collected from eight rabbit foetuses and were isolated by the explant technique. The obtained cells were cultured in DMEM-HIGH glucose and incubated at 37 °C in a humidified atmosphere with 5% CO2.ResultsThe cells adhered to the culture plates and showed a high proliferative capacity with fibroblast-like morphologies. The cells showed a positive response for markers for the cytoskeleton, mesenchymal stem cells and proliferation, pluripotency and haematopoietic precursor stem cells. However, the cells were negative for CD45, a marker of haematopoietic cells. Furthermore, the cells had the capacity to be induced to differentiate into osteogenic, adipogenic and chondrogenic lineages. In addition, when the cells were injected into nude mice, we did not observe the formation of tumours.ConclusionsIn summary, our results demonstrate that multipotent mesenchymal stem cells can be obtained from the rabbit amniotic membrane for possible use in future cell therapy applications.

High-plasticity mesenchymal stem cells isolated from adult-retained primary teeth and autogenous adult tooth pulp—A potential source for regenerative therapies?

PurposeThe objective of this study was to compare the growth rate, morphology, immunohistology and plasticity of autogenous adult-retained SHEDs (arSHEDs) and adult dental pulp stem cells (DPSCs) obtained from the same donor. MethodsExpression of the mesenchymal stem cell markers CD44, CD90, CD105, caspase-3 and GAPDH were assessed using RT-PCR. Caspase-3 and CD44 were also evaluated at the protein level by western blotting of cell lysates. Plasticity of DPSCs and arSHEDs were tested by culture in adipogenic, chondrogenic, osteogenic and Schwann cells induction media. ResultsDPSCs and arSHEDs were isolated by explant culturing and were similarly positive for growth rate and all tested markers. Furthermore, DPSCs and arSHEDs could be driven to adipocyte, chondrocyte, osteocyte and Schwann cells lineages thus indicating similar plasticity as precursor cells. ConclusionThis study demonstrates the similarities between DPSCs and arSHEDs in a unique situation, where both stem cells (SC) types were obtained from a single patient and thus represent an alternative source of SC’s for tissue engineering and regeneration.

Hepatic stem cells and cancers: a pathologist's view.

Neil Theise speaks to Georgia Patey, Commissioning Editor: Neil Theise is a diagnostic liver pathologist, adult stem cell researcher and complexity theorist in New York City, where he is a Professor of Pathology at the Mount Sinai Beth Israel Medical Center of Icahn School of Medicine at Mount Sinai. He received his medical degree from Columbia University College of Physicians and Surgeons, where he also received his training in Anatomic Pathology. Subspecialty training was pursued in gastrointestinal (NYU), liver (Royal Free Hospital) and liver transplant (Mount Sinai, NYC) pathology. His earliest research focus was on defining the premalignant dysplastic nodules in human chronic liver disease. He revised understandings of human liver microanatomy, which in turn, led directly to identification of possible liver stem cell niches and the marrow-to-liver regeneration pathway. He is considered a pioneer of multiorgan adult stem cell plasticity. His publications on these topics in model systems and human liver stem cells have been highlighted on a record five covers of Hepatology.

Switching roles: the functional plasticity of adult tissue stem cells.

Adult organisms have to adapt to survive, and the same is true for their tissues. Rates and types of cell production must be rapidly and reversibly adjusted to meet tissue demands in response to both local and systemic challenges. Recent work reveals how stem cell (SC) populations meet these requirements by switching between functional states tuned to homoeostasis or regeneration. This plasticity extends to differentiating cells, which are capable of reverting to SCs after injury. The concept of the niche, the micro-environment that sustains and regulates stem cells, is broadening, with a new appreciation of the role of physical factors and hormonal signals. Here, we review different functions of SCs, the cellular mechanisms that underlie them and the signals that bias the fate of SCs as they switch between roles.

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice.

Adult stem cells (SCs) reside in niches which balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, SCs outside their niche often display fate flexibility1-4. Here we show that super-enhancers5 underlie the identity, lineage commitment and plasticity of adult SCs in vivo. Using hair follicle (HF) as model, we map the global chromatin domains of HFSCs and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters (‘epicenters’) of transcription factor (TF) binding sites change upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicenters, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, HFSCs dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicenters, enabling them to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of HFSC super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense TF-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status, but also stemness, plasticity in transitional states and differentiation.

Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network.

BackgroundLineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors.ResultsUsing the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other’s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation.ConclusionsOur results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

Segregation of striated and smooth muscle lineages by a Notch-dependent regulatory network

Lineage segregation from multipotent epithelia is a central theme in development and in adult stem cell plasticity. Previously, we demonstrated that striated and smooth muscle cells share a common progenitor within their epithelium of origin, the lateral domain of the somite-derived dermomyotome. However, what controls the segregation of these muscle subtypes remains unknown. We use this in vivo bifurcation of fates as an experimental model to uncover the underlying mechanisms of lineage diversification from bipotent progenitors. Using the strength of spatio-temporally controlled gene missexpression in avian embryos, we report that Notch harbors distinct pro-smooth muscle activities depending on the duration of the signal; short periods prevent striated muscle development and extended periods, through Snail1, promote cell emigration from the dermomyotome towards a smooth muscle fate. Furthermore, we define a Muscle Regulatory Network, consisting of Id2, Id3, FoxC2 and Snail1, which acts in concert to promote smooth muscle by antagonizing the pro-myogenic activities of Myf5 and Pax7, which induce striated muscle fate. Notch and BMP closely regulate the network and reciprocally reinforce each other’s signal. In turn, components of the network strengthen Notch signaling, while Pax7 silences this signaling. These feedbacks augment the robustness and flexibility of the network regulating muscle subtype segregation. Our results demarcate the details of the Muscle Regulatory Network, underlying the segregation of muscle sublineages from the lateral dermomyotome, and exhibit how factors within the network promote the smooth muscle at the expense of the striated muscle fate. This network acts as an exemplar demonstrating how lineage segregation occurs within epithelial primordia by integrating inputs from competing factors.

Differentiation of human dental stem cells reveals a role for microRNA‐218

Regeneration of lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data are available on micro-RNA (miRNA) activity behind human-derived dental stem cells (DSCs). In this study, we isolated periodontal ligament stem cells, dental pulp stem cells and gingival stem cells from extracted third molars; human bone marrow stem cells were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. The miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. The expression of RUNX2 demonstrated successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression that occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human DSCs. DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. These data reveal a miRNA-regulated pathway for the differentiation of human DSCs and a select network of human miRNAs that control DSC osteogenic differentiation.

Enhancing the efficiency of direct reprogramming of human mesenchymal stem cells into mature neuronal-like cells with the combination of small molecule modulators of chromatin modifying enzymes, SMAD signaling and cyclic adenosine monophosphate levels

Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will revolutionize the field of regenerative medicine. While several cell reprogramming methods have been developed over the last decades, the majority of these technologies require the exposure of cell nuclei to reprogramming large molecules via transfection, transduction, cell fusion or nuclear transfer. This raises several technical, safety and ethical issues. Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane penetrable substances to regulate multiple cellular processes including cell plasticity. Recently, using the combination of small molecules that are involved in the regulation chromatin structure and function and agents that favor neural differentiation we have been able to generate neural-like cells from human mesenchymal stem cells. In this study, to improve the efficiency of neuronal differentiation and maturation, two specific inhibitors of SMAD signaling (SMAD1/3 and SMAD3/5/8) that play an important role in neuronal differentiation of embryonic stem cells, were added to our previous neural induction recipe. Results demonstrated that human mesenchymal stem cells grown in this culture conditions exhibited higher expression of several mature neuronal genes, formed synapse-like structures and exerted electrophysiological properties of differentiating neural stem cells. Thus, an efficient method for production of mature neuronal-like cells from human adult bone marrow derived mesenchymal stem cells has been developed. We concluded that specific combinations of small molecules that target specific cell signaling pathways and chromatin modifying enzymes could be a promising approach for manipulation of adult stem cell plasticity.

Spotlight on adipose tissue as a remarkable stem cell source for regenerative medicine and tissue engineering applications

Great scientific strides have been achieved upon discovering the great plasticity of adult mesenchymal stem cells. Such stem cells can be harvested from various sources, including subcutaneous adipose tissue which represents an almost ideal cellular reservoir. Cell therapies using adipose‐derived stem/stromal cells (ASCs) are being conducted in an increasing number of clinical trials such as osteoarthristis and congestive heart failure. The other field reaping the benefits of using ASCs is tissue reconstruction. The multipotency of these cells combined with different engineering strategies result in the in vitro production of a wide variety of human tissues. My research team is using ASCs as building blocks for the production of human tissue substitutes, including adipose tissue itself and skin. They feature a rich extracellular matrix produced by the mesenchymal cells themselves upon ascorbic acid stimulation, recreating a very physiological 3D environment. The reconstructed adipose tissues are functional and secrete important cytokines and growth factors such as leptin, Ang‐1, HGF, and VEGF. This secretome is modulated in a dose‐dependent manner following exposure to the inflammatory cytokine TNFα. Also, production of reconstructed skin using ASCs allowed us to investigate the fate of epithelial stem cells in these engineered tissues, before and after grafting. Supported by CIHR and NSERC.

Advances in cell lineage reprogramming

As a milestone breakthrough of stem cell and regenerative medicine in recent years, somatic cell reprogramming has opened up new applications of regenerative medicine by breaking through the ethical shackles of embryonic stem cells. However, induced pluripotent stem (iPS) cells are prepared with a complicated protocol that results in a low reprogramming rate. To obtain differentiated target cells, iPS cells and embryonic stem cells still need to be induced using step-by-step procedures. The safety of induced target cells from iPS cells is currently a further concerning matter. More broadly conceived is lineage reprogramming that has been investigated since 1987. Adult stem cell plasticity, which triggered interest in stem cell research at the end of the last century, can also be included in the scope of lineage reprogramming. With the promotion of iPS cell research, lineage reprogramming is now considered as one of the most promising fields in regenerative medicine, will hopefully lead to customized, personalized therapeutic options for patients in the future.

Evidence for bone marrow adult stem cell plasticity: properties, molecular mechanisms, negative aspects, and clinical applications of hematopoietic and mesenchymal stem cells transdifferentiation.

In contrast to the pluripotent embryonic stem cells (ESCs) which are able to give rise to all cell types of the body, mammalian adult stem cells (ASCs) appeared to be more limited in their differentiation potential and to be committed to their tissue of origin. Recently, surprising new findings have contradicted central dogmas of commitment of ASCs by showing their plasticity to differentiate across tissue lineage boundaries, irrespective of classical germ layer designations. The present paper supports the plasticity of the bone marrow stem cells (BMSCs), bringing the most striking and the latest evidences of the transdifferentiation properties of the bone marrow hematopoietic and mesenchymal stem cells (BMHSCs, and BMMSCs), the two BM populations of ASCs better characterized. In addition, we report the possible mechanisms that may explain these events, outlining the clinical importance of these phenomena and the relative problems.

Breastmilk is a novel source of stem cells with multilineage differentiation potential.

The mammary gland undergoes significant remodeling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self-renewal in breast cancer. Here, we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4, SOX2, NANOG, known to constitute the core self-renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts, a population of hBSCs exhibited an encapsulated ESC-like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self-renewal TFs were found silenced in the normal resting epithelium, they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore, hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient-specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer, with potential use in bioengineering and tissue regeneration. Stem Cells2012;30:2164–2174

Adult stem cell plasticity: Dream or reality?

A stem cell is a primitive cell that can either divide to reproduce itself (undergo self renewal) or give rise to more specialized (differentiated) cells. Stem cells exist in many adult tissues. Stem cell exists in a variety of organs in a variety of phenotypes, in greater plenitude than previously suspected. By contrast, embryonic stem cells are derived from an embryonic cell population at a stage prior to its commitment to form particular tissues of the body. Because of their origin, embryonic stem cells are by nature more versatile than most of their adult counterparts. The first cell commitment event in the mammalian embryo occurs with compaction of the embryo and formation of the outer trophoblast layer. The small cluster of cells inside the blastocyst, the inner cell mass, is destined to give rise to all the tissues of the body. It is at this stage of development that stem cells are isolated. Human embryonic stem cell lines are made by dissecting an early blastocyst- stage embryo and removing its inner cell mass. The concept of stem cell has changed greatly in recent years. The potential for stem cells to regenerate non-haematopoietic tissues, offers an expanded repertoire of clinical applications for stem cell transplantation. The presumed ability of tissue specific stem cells to acquire the fate of cell types different from the tissue of origin has been termed adult stem cell plasticity. Since a series of papers heralded the finding of surprising plasticity in a variety of adult stem cells [1, 2], this exciting field has generated almost as much controversy and rhetoric as it has scientific insights. This brief review will attempt to highlight some of the concepts and concerns regarding adult cell plasticity.

Dedifferentiating Spermatogonia Outcompete Somatic Stem Cells for Niche Occupancy in the Drosophila Testis

Differentiating cells can dedifferentiate to replace stem cells in aged or damaged tissues, but the underlying mechanisms are unknown. In the Drosophila testis, a cluster of stromal cells called the hub creates a niche by locally activating Janus kinase-signal transducer and activator of transcription (Jak-STAT) signaling in adjacent germline and somatic stem cells. Here, we establish a system to study spermatogonial dedifferentiation. Ectopically expressing the differentiation factor bag-of-marbles (Bam) removes germline stem cells from the niche. However, withdrawing ectopic Bam causes interconnected spermatogonia to fragment, move into the niche, exchange positions with resident somatic stem cells, and establish contact with the hub. Concomitantly, actin-based protrusions appear on subsets of spermatogonia, suggesting acquired motility. Furthermore, global downregulation of Jak-STAT signaling inhibits dedifferentiation, indicating that normal levels of pathway activation are required to promote movement of spermatogonia into the niche during dedifferentiation, where they outcompete somatic stem cells for niche occupancy.

Environmental Changes Impair Stem and Progenitor Cell Function and Contribute to Cardiovascular Aging

Cardiac stem and progenitor cells have been identified in the heart, bone marrow and other organs and have been shown to contribute to cardiovascular repair via both direct replacement and paracrine signaling that promotes local regeneration. These cells are maintained through adulthood and are present even in older adults. This finding is notable in light of the reduced capacity for repair and regeneration in older tissues. This apparent dichotomy can be reconciled through an appreciation of the age‐associated changes in the microenvironmental pathways that govern adult stem cell plasticity and function. Besides intrinsic changes to the cells themselves that are associated with aging, including DNA damage and loss of telomeres, changes in the microenvironment of the cell, both in the stem cell niche and at the site of injury, may impact the regenerative ability of the aging heart. We have identified a number of environmental pathways that become dysregulated over time and may underlie the impairments in cardiac stem and progenitor cell function with increasing age. It is hypothesized that identification of mechanisms to reverse these age‐associated changes may lead to the development of novel therapeutic strategies to enhance stem‐ and progenitor cell‐mediated repair and promote cardiovascular health in older individuals.