The in vivo developmental potential of porcine skin-derived progenitors and neural stem cells

Multipotent skin-derived progenitors (SKPs) can be traced back to embryonic neural crest cells and are able to differentiate into both neural and mesodermal progeny in vitro. Neural stem cells (NSCs) are capable of self-renewing and can contribute to neuron and glia in the nervous system. Recently, we derived porcine SKPs and NSCs from the same enhanced green fluorescent protein (EGFP) transgenic fetuses and demonstrated that SKPs could contribute to neural and mesodermal lineages in vivo. However, it remains unclear whether porcine SKPs and NSCs can generate ectoderm and mesoderm lineages or other germ layers in vivo. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus, the purpose of this study was to investigate the in vivo developmental potential of porcine SKPs and fetal brain-derived NSCs by chimera production. Porcine SKPs, NSCs, and fibroblasts were injected into precompact in vitro fertilized embryos (IVF) and then transferred into corresponding surrogates 24?h postinjection. We found that porcine SKPs could incorporate into the early embryos and contribute to various somatic tissues of the 3 germ layers in postnatal chimera, and especially have an endodermal potency. However, this developmental potential is compromised when they differentiate into fibroblasts. In addition, porcine NSCs fail to incorporate into host embryos and contribute to chimeric piglets. Therefore, neural crest-derived SKPs may represent a more primitive state than their counterpart neural stem cells in terms of their contributions to multiple cell lineages.     

The potential of mouse skin-derived precursors to differentiate into mesenchymal and neural lineages and their application to osteogenic induction in vivo

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.     

Human embryonic stem cells: lessons from stem cell niches in vivo

In vivo the stem cell niche is an essential component in controlling and maintaining the stem cells' ability to survive and respond to injury. Human embryonic stem cells (hESCs) appear to be an exception to this rule as they can be removed from their blastocytic microenvironment and maintained indefinitely in vitro. However, recent observations reveal the existence of an autonomously derived in vitro hESC niche. This provides a previously unappreciated mechanism to control hESC expansion and differentiation. Recognizing this, it may now be possible to take aspects of in vivo stem cell niches, namely extracellular matrices, paracrine signals and accessory cell types, and exploit them in order to gain fidelity in directed hESC differentiation. In doing so, routine customization of hESC lines and their application in regenerative therapies may be further enhanced using unique hESC niche-based approaches.     

Regulation of hepatic stem/progenitor phenotype by microenvironment stiffness in hydrogel models of the human liver stem cell niche

Human livers have maturational lineages of cells within liver acini, beginning periportally in stem cell niches, the canals of Hering, and ending in polyploid hepatocytes pericentrally and cholangiocytes in bile ducts. Hepatic stem cells (hHpSCs) in?vivo are partnered with mesenchymal precursors to endothelia (angioblasts) and stellate cells, and reside in regulated microenvironments, stem cell niches, containing hyaluronans (HA). The in?vivo hHpSC niche is modeled in?vitro by growing hHpSC in two-dimensional (2D) cultures on plastic. We investigated effects of 3D microenvironments, mimicking the liver's stem cell niche, on these hHpSCs by embedding them in HA-based hydrogels prepared with Kubota's Medium (KM), a serum-free medium tailored for endodermal stem/progenitors. The KM-HA hydrogels mimicked the niches, matched diffusivity of culture medium, exhibited shear thinning and perfect elasticity under mechanical loading, and had predictable stiffness depending on their chemistry. KM-HA hydrogels, which supported cell attachment, survival and expansion of hHpSC colonies, induced transition of hHpSC colonies towards stable heterogeneous populations of hepatic progenitors depending on KM-HA hydrogel stiffness, as shown by both their gene and protein expression profile. These acquired phenotypes did not show morphological evidence of fibrotic responses. In conclusion, this study shows that the mechanical properties of the microenvironment can regulate differentiation in endodermal stem cell populations.     

The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels

Synthetic hydrogel scaffolds that can be used as culture systems that mimic the natural stem cell niche are of increased importance for stem cell biology and regenerative medicine. These artificial niches can be utilized to control the stem cell fate and will have potential applications for expanding/differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. In this study, we synthesized hyaluronic acid (HA) hydrogels that could be degraded through a combination of cell-released enzymes and used them to culture mouse mesenchymal stem cells (mMSC). To form the hydrogels, HA was modified to contain acrylate groups and crosslinked through Michael addition chemistry using non-degradable, plasmin degradable or matrix metalloproteinase (MMP) degradable crosslinkers. Using this hydrogel we found that mMSC proliferation occurred in the absence of cell spreading, that mMSCs could only spread when both RGD and MMP degradation sites were present in the hydrogel and that mMSCs in hydrogels with high density of RGD (1000 μm) spread and migrated faster and more extensively than in hydrogels with low density of RGD (100 μm).     

Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels,Matrigel and Collagen I

Designer peptides with self-assembling properties form nanofibers which are further organized to form a hydrogel consisting of up to 99.5% water. We present here the encapsulation of neural stem cells into peptide nanofiber hydrogel scaffolds. This results in three-dimensional (3-D) neural tissue cultures in which neural stem cells differentiate into progenitor neural cells, neurons, astrocytes and oligodendrocytes when cultured in serum-free medium. Cell survival studies showed that neural cells in peptide hydrogels thrive for at least 5 months. In contrast, neural stem cells encapsulated in Collagen I were poorly differentiated and did not migrate significantly, thus forming clusters. We show that for culture periods of 1–2 weeks, neural stem cells proliferate and differentiate better in Matrigel. However, in long-term studies, the population of cells in Matrigel decreases whereas better cell survival rates are observed in neural tissue cultures in peptide hydrogels. Peptide functionalization with cell adhesion and cell differentiation motifs show superior cell survival and differentiation properties compared to those observed upon culturing neural cells in non-modified peptide hydrogels. These designed 3-D engineered tissue culturing systems have a potential use as tissue surrogates for tissue regeneration. The well-defined chemical and physical properties of the peptide nanofiber hydrogels and the use of serum-free medium allow for more realistic biological studies of neural cells in a biomimetic 3-D environment.     

体外构建胚胎干细胞生长分化的三维研究模型

目的以液态胶原为支架,小鼠胚胎干细胞(ESCs)为细胞模型,构建ESCs胶-原复合体,旨在尝试建立一种能够实现干细胞生长、分化的三维培养体系。方法提取鼠尾胶原,观察ESCs在自制胶原条带内增殖的形态特征,并测定葡萄糖/乳酸活性。以ESCs源心肌细胞为目的细胞,利用免疫组化、RT-PCR及电镜技术评价胶原条带内ESCs进行自发性分化的能力。结果ESCs在胶原条带所提供的三维培养体系内生长、增殖状态良好,且彼此间能够建立细胞连接。胶原条带内部分ESCs能够自发性分化为心肌细胞。该心肌细胞均可表达心肌蛋白cTn-T,心肌转录因子NKX2.5及肌球蛋白轻链MLC-2 vmRNA,且肌小节结构发育成熟。结论以液态Ⅰ型胶原为主体的支架材料可以为ESCs提供良好的生长基质,促进其组织化发育。该实验初步明确了体外构建干细胞生长分化三维模型的可行性。     

The endothelial cell line bEnd.3 maintains human pluripotent stem cells

Endothelial cells line blood vessels and coordinate many aspects of vascular biology. More recent work has shown that endothelial cells provide a key niche in vivo for neural stem cells. In vitro, endothelial cells secrete a factor that expands neural stem cells while inhibiting their differentiation. Here, we show that a transformed mouse endothelial cell line (bEnd.3) maintains human pluripotent stem cells in an undifferentiated state. bEnd.3 cells have a practical advantage over mouse embryonic fibroblasts for pluripotent stem cell maintenance since they can be expanded in vitro and engineered to express genes of interest. We demonstrate this capability by producing fluorescent and drug-resistant feeder cells. Further, we show that bEnd.3 secretes an activity that maintains human embryonic stem cells without direct contact.     

Basement Membrane Matrix (BME) has Multiple Uses with Stem Cells

The utilization of basement membrane matrix has helped to overcome many of the obstacles associated with stem cell research. Initially, there were several problems with investigating stem cells, including difficult extraction from tissues, the need for feeder layers, poor survival, minimal proliferation, limited differentiation in vitro, and inadequate survival when injected or transplanted in vivo. Given that the basement membrane is the first extracellular matrix that is produced by the developing embryo, it was quickly identified as an important factor for modulating stem cell behavior, and since then, basement membrane extract (BME) has been successfully employed in numerous methods as a substratum in vitro and as a bioactive support in vivo to overcome many of these problems. A thin BME coating is sufficient to maintain an undifferentiated phenotype during embryonic stem cell expansion, while a thick BME hydrogel may be employed to induce stem cell differentiation. BME also promotes stem cell survival for in vivo applications and provides a physiological environment for evaluating stem cell co-culture with other cell types. The present article provides a concise review of current methodologies utilizing BME for stem cell research.     

In vivo ectopic chondrogenesis of BMSCs directed by mature chondrocytes

In vivo niche plays an important role in determining the fate of exogenously implanted stem cells. Due to the lack of a proper chondrogenic niche, stable ectopic chondrogenesis of mesenchymal stem cells (MSCs) in subcutaneous environments remains a great challenge. The clinical application of MSC-regenerated cartilage in repairing defects in subcutaneous cartilage such as nasal or auricular cartilage is thus severely limited. The creation of a chondrogenic niche in subcutaneous environments is the key to solving this problem. The current study demonstrates that bone marrow stromal cells (BMSCs) could form cartilage-like tissue in a subcutaneous environment when co-transplanted with articular chondrocytes, indicating that chondrocytes could create a chondrogenic niche to direct chondrogenesis of BMSCs. Then, a series of in vitro co-culture models revealed that it was the secretion of soluble factors by chondrocytes but not cell-cell contact that provided the chondrogenic signals. The subsequent studies further demonstrated that multiple factors currently used for chondroinduction (including TGF-β1, IGF-1 and BMP-2) were present in the supernatant of chondrocyte-engineered constructs. Furthermore, all of these factors were required for initiating chondrogenic differentiation and fulfilled their roles in a coordinated way. These results suggest that paracrine signaling of soluble chondrogenic factors provided by chondrocytes was an important mechanism in directing the in vivo ectopic chondrogenesis of BMSCs. The multiple co-culture systems established in this study provide new methods for directing committed differentiation of stem cells as well as new in vitro models for studying differentiation mechanism of stem cells determined by a tissue-specific niche.     

Chondrogenic potential of stem cells derived from amniotic fluid, adipose tissue, or bone marrow encapsulated in fibrin gels containing TGF-β3

In this study, several types of hMSCs, derived from bone marrow, adipose tissue, or amniotic fluid, were encapsulated in a fibrin hydrogel mixed with TGF-β3 and then evaluated for their capacity for differentiation in?vitro and in?vivo. For determination of stem cell differentiation, RT-PCR, real time quantitative PCR (qPCR), histology, and immunohistochemical assays were used for analysis of chondrogenesis. Using these analysis methods, several of the cultured hMSCS were found to highly express genes and proteins specific to cartilage forming tissues. Additionally, similar trends in expression were found in tissue recovered from nude mice transplanted with several types of hMSCs encapsulated in a fibrin hydrogel containing TGF-β3. The results of both in?vitro and in?vivo analyses showed that cultured or transplanted hMSCs mixed with TGF-β3 in a fibrin hydrogel differentiated into chondrocytes, suggesting that these cells would be suitable for reconstruction of hyaline articular cartilage.     

Skin-derived Precursors as a Source of Progenitors for Cutaneous Nerve Regeneration

Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain, and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth-permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells dedifferentiate to a progenitor/stem cell phenotype to promote axonal regrowth. In this study, we show that skin-derived precursors (SKPs), a recently identified neural crest-related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro three-dimensional cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis. STEM Cells2012;30:2261-2270.     

A highly enriched niche of precursor cells with neuronal and glial potential within the hair follicle dermal papilla of adult skin

Skin-derived precursor cells (SKPs) are multipotent neural crest-related stem cells that grow as self-renewing spheres and are capable of generating neurons and myelinating glial cells. SKPs are of clinical interest because they are accessible and potentially autologous. However, although spheres can be readily isolated from embryonic and neonatal skin, SKP frequency falls away sharply in adulthood, and primary sphere generation from adult human skin is more problematic. In addition, the culture-initiating cell population is undefined and heterogeneous, limiting experimental studies addressing important aspects of these cells such as the behavior of endogenous precursors in vivo and the molecular mechanisms of neural generation. Using a combined fate-mapping and microdissection approach, we identified and characterized a highly enriched niche of neural crest-derived sphere-forming cells within the dermal papilla of the hair follicle of adult skin. We demonstrated that the dermal papilla of the rodent vibrissal follicle is 1,000-fold enriched for sphere-forming neural crest-derived cells compared with whole facial skin. These "papillaspheres" share a phenotypic and developmental profile similar to that of SKPs, can be readily expanded in vitro, and are able to generate both neuronal and glial cells in response to appropriate cues. We demonstrate that papillaspheres can be efficiently generated and expanded from adult human facial skin by microdissection of a single hair follicle. This strategy of targeting a highly enriched niche of sphere-forming cells provides a novel and efficient method for generating neuronal and glial cells from an accessible adult somatic source that is both defined and minimally invasive.     

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.     

Toward modeling the bone marrow niche using scaffold-based 3D culture systems

In the bone marrow, specialized microenvironments, called niches, regulate hematopoietic stem cell (HSC) maintenance and function through a complex crosstalk between different cell types. Although in vivo studies have been instrumental to elucidate some of the mechanisms by which niches exert their function, the establishment of an in vitro model that recapitulates the fundamental interactions of the niche components in a controlled setting would be of great benefit. We have previously shown that freshly harvested bone marrow- or adipose tissue-derived cells can be cultured under perfusion within porous scaffolds, allowing the formation of an organized 3D stromal tissue, composed by mesenchymal and endothelial progenitors and able to support hematopoiesis. Here we describe 3D scaffold-based perfusion systems as potential models to reconstruct ex vivo the bone marrow stem cell niche. We discuss how several culture parameters, including scaffold properties, cellular makeup and molecular signals, can be varied and controlled to investigate the role of specific cues in affecting HSC fate. We then provide a perspective of how the system could be exploited to improve stem cell-based therapies and how the model can be extended toward the engineering of other specialized stromal niches.     

Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression

In normal hematopoiesis, proliferation is tightly linked to differentiation in ways that involve cell-cell interaction with stromal elements in the bone marrow stem cell niche. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell renewal and hematopoiesis. Not surprisingly, mutations in the Notch gene have been linked to a number of types of malignancies. To better define the function of Notch in both normal and neoplastic hematopoiesis, a tetracycline-inducible system regulating expression of a ligand-independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. During coculture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic stem cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their survival and proliferative capacity and lowers the levels of hematopoietic stem cell markers CD34 and c-Kit and the myeloid marker CD11b. Global gene expression profiling of day 8 hematopoietic progenitors in the absence and presence of activated Notch yield candidate genes required for normal hematopoietic differentiation, as well as putative downstream targets of oncogenic forms of Notch including the noncanonical Wnts Wnt4 and 5A. Disclosure of potential conflicts of interest is found at the end of this article.     

An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells

It is controversial whether a biomaterial itself, rather than addition of any exogenous growth factor, could induce mesenchymal stem cells (MSCs) to differentiate into chondrogenic lineage, further to regenerate cartilage. Previous studies have shown that collagen-based hydrogel could induce MSCs to differentiate into chondrocytes in vivo but the in vitro studies only have a few reports. The evidence that biomaterials could induce chondrogenesis is not adequate. In this study, we tried to address whether type I collagen hydrogel has chondro-inductive capability in vitro and how this scaffold induces MSCs to generate cartilage tissue without exogenous growth factors in the culture medium. We encapsulated neonatal rabbit bone marrow mesenchymal stem cells (BMSCs) in type I collagen hydrogel homogeneously or implanted cell aggregates in hydrogel, and cultured them in nonchondrogenic inductive media. After at least 28 days culture, cells in the homogeneous group were tending to chondrogenic differentiation while cell density was high, and cells in the aggregate group have almost gone through chondrogenesis and formed neo-cartilage tissue with abundant specific extracellular matrix (ECM) deposition. These results indicate collagen hydrogel has inherent inductivity for the chondrogenic differentiation of BMSCs, and the optimum specification and tissue formation were accompanied with local high cell density. This research suggests a feasible strategy to induce the chondro differentiation of BMSCs independent of exogenous growth factors, which may greatly contribute to clinical cartilage regeneration. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2717-2725, 2012.     

Adult hair follicle stem cells do not retain the older DNA strands in vivo during normal tissue homeostasis

Tissue stem cells have been proposed to segregate the chromosomes asymmetrically (in a non-random manner), thereby retaining preferentially the older “immortal” DNA strands bearing the stemness characteristics into one daughter cell, whereas the newly synthesized strands are segregated to the other daughter cell that will commit to differentiation. Moreover, this non-random segregation would protect the stem cell genome from accumulating multiple mutations during repeated DNA replication. This long-standing hypothesis remains an active subject of study due to conflicting results for some systems and lack of consistency among different tissue stem cell populations. In this review, we will focus on work done in the hair follicle, which is one of the best-understood vertebrate tissue stem cell system to date. In cell culture analysis of paired cultured keratinocytes derived from hair follicle, stem cells suggested a non-random segregation of chromosome with respect to the older DNA strand. In vivo, the hair follicle stem cells appear to self-renew and differentiate at different phases of their homeostatic cycle. The fate decisions occur in quiescence when some stem cells migrate out of their niche and commit to differentiation without self-renewal. The stem cells left behind in the niche self-renew symmetrically and randomly segregate the chromosomes at each division, making more stem cells. This model seems to apply to at least a few other vertebrate tissue stem cells in vivo.     

Engineering interpenetrating network hydrogels as biomimetic cell niche with independently tunable biochemical and mechanical properties

Hydrogels have been widely used as artificial cell niche to mimic extracellular matrix with tunable properties. However, changing biochemical cues in hydrogels developed-to-date would often induce simultaneous changes in mechanical properties, which do not support mechanistic studies on stem cell-niche interactions. Here we report the development of a PEG-based interpenetrating network (IPN), which is composed of two polymer networks that can independently and simultaneously crosslink to form hydrogels in a cell-friendly manner. The resulting IPN hydrogel allows independently tunable biochemical and mechanical properties, as well as stable and more homogeneous presentation of biochemical ligands in 3D than currently available methods. We demonstrate the potential of our IPN platform for elucidating stem cell-niche interactions by modulating osteogenic differentiation of human adipose-derived stem cells. The versatility of such IPN hydrogels is further demonstrated using three distinct and widely used polymers to form the mechanical network while keeping the biochemical network constant.