Two- and Three-Dimensional Culture of Keratinocyte Stem and Precursor Cells Derived from Primary Murine Epidermal Cultures

In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.     

Functional and phenotypic characterization of human keratinocytes expanded in microcarrier culture

Skin cells for transplantation are routinely prepared by growing patient keratinocytes in a semi-defined cocktail of growth factors, including serum and feeder cells. However, these reagents require substantial risk remediation and can contribute to transplant rejection. Microcarrier culture is an emerging technology that may allow the elimination of feeder cells whilst facilitating expansion of cultured keratinocytes. However, the behavior of keratinocytes in microcarrier culture and the potential of these cells to form an epidermis have been poorly defined. We characterized freshly isolated human keratinocytes cultured on CultiSpher-G microcarriers in the absence of murine feeder cells and assessed the potential of the keratinocytes to form an epidermis in an in vitro model. In a single passage, keratinocytes multiplied 44.9-fold in microcarrier-bioreactor culture in 17 days, whereas two-dimensional cultures reached confluence in 9 days and only expanded 7.4-fold. Histological characterization of keratinocytes on the microcarriers revealed that the cells were randomly distributed within these porous structures, however, not all pores contained cells. High-resolution microcomputed tomography imaging of the microcarriers confirmed limited interconnectivity of the pores. Immunoreactivity of specific epidermal markers was confirmed during cell expansion via immunohistochemistry. Despite the expression of differentiation markers, microcarrier-expanded keratinocytes retained the capacity to form an epidermis, as was evaluated using an in vitro human skin equivalent model. The epidermis formed by microcarrier-expanded keratinocytes in this model exhibited morphology similar to native skin. Significantly, the microcarrier technique successfully eliminates the need for a feeder cell layer and hence facilitates development of an improved culture system.     

Generation of human epidermal constructs on a collagen layer alone

Because engineered tissues are designed for clinical applications in humans, a major problem is the contamination of cocultures and tissues by allogenic molecules used to grow stem cells in vitro. The protocols that are commonly applied to generate epidermal equivalents in vitro require the use of irradiated murine fibroblasts as a feeder layer for keratinocytes. In this study, we report a simple procedure for growing human keratinocytes, isolated from adult skin, to generate an epidermal construct on a collagen layer alone. In this model, no human or murine feeder layers were used to amplify cell growth, and isolated keratinocytes were seeded directly at high cell density on the collagen-coated flasks or coverslips in an epithelial growth medium containing low calcium concentration. Morphological, immunochemical, and cytokinetic features of epithelial colonies grown on the collagen layer were typical of keratinocytes and were comparable with those reported for keratinocytes grown on a feeder layer. The stratification of keratinocytes generated 3-dimensional synthetic constructs displaying a tissue architecture comparable with that of natural epidermis. Epithelial cells expressed specific markers of keratinocyte terminal differentiation, including involucrin and filaggrin. Nevertheless, the number of cell layers was lower than in natural skin, and electron microscopical analysis revealed that the overall organization of these layers was poor compared with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization. The lack of epithelial-mesenchymal interactions that occur during skin histogenesis may account for such an incomplete maturation of epidermal constructs.     

A new isolation method of human limbal progenitor cells by maintaining close association with their niche cells

In human corneal epithelium, self-renewal and fate decision of stem cells are highly regulated in a niche microenvironment called palisades of Vogt in the limbus. Herein, we discovered that digestion with dispase, which cleaves off the basement membrane, did not remove the entire basal epithelial progenitor cells. In contrast, digestion with collagenase isolated on cluster consisting of not only entire epithelial progenitor cells but also their closely associated mesenchymal cells because of better preservation of some basement membrane matrix. Collagenase isolated more basal epithelial progenitor cells, which were p63α+ and small in the size (8 μm in diameter), and generated significantly more holoclones and meroclones on 3T3 fibroblast feeder layers than dispase. Further, collagenase isolated more small pan-cytokeratin-/p63α-/vimentin+ cells with the size as small as 5 μm in diameter and heterogeneously expressing vimentin, Oct4, Sox2, Nanog, Rex1, Nestin, N-cadherin, SSEA4, and CD34. Maintenance of close association between them led to clonal growth in a serum-free, low-calcium medium, whereas disruption of such association by trypsin/EDTA resulted in no clonal growth unless cocultured with 3T3 fibroblast feeder layers. Similarly, on epithelially denuded amniotic membrane, maintenance of such association led to consistent and robust epithelial outgrowth, which was also abolished by trypsin/EDTA. Epithelial outgrowth generated by collagenase-isolated clusters was significantly larger in diameter and its single cells yielded more holoclones on 3T3 fibroblast feeder layers than that from dispase-isolated sheets. This new isolation method can be used for exploring how limbal epithelial stem cells are regulated by their native niche cells.     

Mannosides as crucial part of bioactive supports for cultivation of human epidermal keratinocytes without feeder cells

Large-scale production of keratinocytes by cell culture is of interest for medical applications. Long-term cultivation of epidermal cells is presently possible with feeder cells, i.e. 3T3 fibroblasts with arrested mitosis, or with specially formulated culture medium. To define refinements for in vitro conditions, the analysis of the natural environment with growth-maintaining/stimulating factors can provide important clues. Cells with proliferative activity are located in the basal layer of the epidermis in close contact with a basement membrane. Employing lectin and reverse lectin histochemistry of skin, muscle fibers and feeder cells, we assumed that the interplay of mannose-binding sites of epidermal cells, detected by a labeled neoglycoprotein, with glycoligands in the feeder cell layer or basement membrane could trigger signaling with relevance for adhesion and growth regulation. Indeed, coating of polystyrene with mannose-containing neoglycoprotein mimicking a mannose-rich cell matrix enabled the cultivation of keratinocytes without feeder cells in a Ca(2+)-dependent manner in serum-containing culture medium. Following this experimental demonstration of specific binding of mannose residues as part of a neoglycoprotein controlled by testing sugar-free carrier protein and other substances, we next synthesized and tested biocompatible polymers. Attachment and proliferation of keratinocytes on the surface of these polymers compared favorably to control experiments using feeder cells. In conclusion, we suggest that these polymers are bioactive offering a perspective for keratinocyte cultivation without feeder cells.     

Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells

Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.     

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.     

Human hepatoblast phenotype maintained by hyaluronan hydrogels

Human hepatoblasts and hepatic stem cells, pluripotent hepatic progenitors that give rise to hepatocytes and biliary cells, were isolated from fetal livers and found to express hyaluronan receptors (CD44) in both the freshly isolated cells and after culture. This implicates an in vivo connection to hyaluronan (HA), an embryonic matrix component, as a candidate 3-dimensional (3-D) scaffold for hepatic progenitor cell expansion and/or differentiation. To assess HAs as scaffolds, hepatoblasts and hepatic stem cells were seeded into HA hydrogels with a serum-free, hormonally defined medium tailored for expansion of hepatic progenitors. Cell aggregates formed within the HA hydrogels and remained viable, proliferative, and demonstrated a stable phenotype intermediate between that of hepatic stem cells and hepatoblasts throughout more than 4 weeks of culturing, with little evidence of lineage restriction towards either hepatocytic or biliary pathways. The phenotype consisted of stable co-expression of both hepatocytic and biliary markers such as biliary-specific cytokeratin, CK19, low levels of expression of albumin, and urea synthesis. HA hydrogels are ideal as 3-D scaffolds for pluripotent hepatic progenitors and should be useful for generating cells to be used in bioartificial livers or tissue engineered liver grafts.     

Innate immunity mediated by epidermal keratinocytes promotes acquired immunity involving Langerhans cells and T cells in the skin

Skin is an immunological organ consisting of epidermal cells, i.e. keratinocytes and Langerhans cells (LCs, antigen-presenting dendritic cells), and both innate and acquired immune systems operate upon exposure of the skin to various external microbes or their elements. To explore the relationship between innate and acquired immunities in the skin, we investigated whether Toll-like receptor (TLR) ligation of epidermal cells enhances the ability of LCs to present a specific antigen to T cells in mice. LC-containing epidermal cells were incubated with CpG oligonucleotide (TLR9 ligand) modified with trinitrophenyl hapten, and cultured with hapten-primed CD4(+) T cells. TLR9 ligand was capable of enhancing the hapten-presenting ability of LCs when LC-enriched epidermal cells, but not purified LCs, were used as the LC source, suggesting that bystander keratinocytes play a role in the enhancement of LC function. Cultivation of freshly isolated epidermal cells with CpG promoted the expression of major histocompatibility complex (MHC) class II and CD86 molecules on LCs. CpG enhanced the production of interleukin (IL)-1alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha by primarily cultured keratinocytes. The addition of a cocktail of neutralizing antibodies against these cytokines abrogated the CpG-promoted, antigen-presenting ability of LC-enriched epidermal cells. Moreover, the addition of culture supernatants from CpG-stimulated keratinocytes restored the ability of purified LCs. Our study demonstrated that although the direct effect of CpG on LCs is minimal, LC function can be up-regulated indirectly by cytokines released by CpG-stimulated keratinocytes. This also implies that innate immunity evoked by TLR ligation of keratinocytes enhances acquired immunity comprising LCs and T cells.     

表皮干细胞在组织工程创面修复中的应用

背景：随着细胞生物学、分子生物学技术、组织工程学的快速发展,探寻组织工程化皮肤创面覆盖物的“种子细胞”的研究逐渐增多。 目的：总结表皮干细胞的生物学特性,探讨其在皮肤创面修复过程中的再生作用与临床应用价值。 方法：应用计算机检索CNKI和PubMed数据库中2002-07/2011-12关于表皮干细胞修复皮肤损伤研究的文章,以“表皮干细胞,创面修复,组织工程,皮肤”或“epidermal stem cells,tissue engineering skin,wound surface”为检索词进行检索。选择文章内容与表皮干细胞修复皮肤损伤研究进展有关的文章,同一领域文献则选择近期发表或发表在权威杂志文章。初检得到129篇文献,根据纳入标准选择28篇文献进行综合分析。 结果与结论：表皮干细胞是表皮发生、分化和创面修复的基础,其正常增殖分化是维持皮肤正常组织结构和细胞内环境稳定的基本要求,也是皮肤组织工程理想的种子细胞。对烧伤、创伤等大面积皮肤缺损的治疗,对皮肤疾病的细胞疗法、基因治疗等方面都有很好的应用前景。表皮细胞的体外培养是复合人工皮肤组织工程学研究的先决条件。随着对表皮干细胞分离、纯化和培养技术的不断完善,可达到迅速构建表皮层的目的。但表皮干细胞的应用研究仍需要进一步的探索。     

Mixture of fibroblasts and adipose tissue-derived stem cells can improve epidermal morphogenesis of tissue-engineered skin

Many studies demonstrate that the type of adjacent mesenchymal cells can affect epidermal morphogenesis of bilayered tissue-engineered skin. However, whether a mixture of different mesenchymal cell types can improve epidermal morphogenesis of bioengineered skin remains unknown. In this study, keratinocytes, dermal fibroblasts and adipose tissue-derived stem cells (ADSCs) were isolated and purified from human skin and subcutaneous fat. Conditioned medium generated from a mixture of dermal fibroblasts and ADSCs at the ratio of 1:1 was superior to that from fibroblasts or ADSCs alone in promoting keratinocyte proliferation, as indicated by MTT assay. Furthermore, ELISA results showed that the cytokine levels of human hepatocyte growth factor and keratinocyte growth factor (also known as FGF7) in the mixed fibroblasts/ADSC group were higher than those in the ADSC or dermal fibroblasts group. To examine the potential roles of mixed fibroblasts and ADSCs on epidermal morphogenesis, a three-dimensional tissue engineered skin system was applied. Histological analyses demonstrated that keratinocytes proliferated extensively over the mixture of fibroblasts and ADSCs, and formed a thick epidermal layer with well-differentiated structures. Keratin 10 (epidermal differentiation marker) was expressed in the suprabasal layer of bilayered tissue-engineered skin in the mixed fibroblasts and ADSCs group. Desmosomes and hemidesmosomes were detected in the newly formed epidermis by transmission electron microscopy analysis. Together, these findings revealed for the first time that a mixture of fibroblasts and ADSCs in bilayered tissue-engineered skin can improve epidermal morphogenesis.     

Maintenance of proliferative capacity and retroviral transduction efficiency of human fetal CD38(-)/CD34(++) stem cells

Methods for the efficient transduction and expansion of fetal hematopoietic stem cells could lead to novel in utero therapies for blood cell disorders and enzymatic deficiencies. Here we describe a new assay to measure rapidly the effects of cytokines on the differentiation or expansion of primitive progenitors and stem cells found among CD38(-)CD34(++) lineage() cells isolated from human midgestation liver. Importantly, conditions that otherwise supported the expansion of clonogenic progenitors reduced their proliferative capacity. A combination of megakaryocyte growth and development factor and granulocyte-macrophage colony-stimulating factor maintained proliferative potential while also yielding an intermediate level of progenitor expansion. Retroviral transduction was achieved using Moloney murine leukemia virus-based vectors. Freshly isolated candidate stem cells could be transduced at almost 17% efficiency by a 1-h exposure to virus with centrifugation to aid transduction. This was increased to a mean 35.5% transduction efficiency after 1 day of culture. Additionally, the transduction efficiency of candidate stem cells isolated from fetal placental blood was 33.0%. These findings encourage further investigation into the feasibility of ex utero gene therapy whereby fetal cells are isolated from the circulation, transduced, and expanded ex utero before being returned to the fetus.     

Functional characterization of quiescent keratinocyte stem cells and their progeny reveals a hierarchical organization in human skin epidermis

Although homeostatic renewal of human skin epidermis is achieved by the combined activity of quiescent stem cells (SCs) and their actively cycling progeny, whether these two populations are equipotent in their capacity to regenerate tissue has not been determined in biological assays that mimic lifelong renewal. Using fluorescence activated cell separation strategy validated previously by us, human epidermis was fractionated into three distinct subsets: that is, α?6briCD71(dim) , α?6briCD71(bri) , and α?6dim with characteristics of keratinocyte stem, transient amplifying, and early differentiating cells, respectively. The global gene expression profile of these fractions was determined by microarray, confirming that the α?6briCD71(dim) subset was quiescent, the α?6briCD71(bri) was actively cycling, and the α?6dim subset expressed markers of differentiation. More importantly, functional evaluation of these populations in an in vivo model for tissue reconstitution at limiting cell dilutions revealed that the quiescent α?6briCD71(dim) fraction was the most potent proliferative and tissue regenerative population of the epidermis, capable of long-term (LT) epidermal renewal from as little as 100 cells for up to 10 weeks. In contrast, the cycling α?6briCD71(bri) fraction was the first to initiate tissue reconstitution, although this was not sustained in the LT, while differentiating α?6dim cells possessed the lowest demonstrable tissue regenerative capacity. Our data suggest that in human skin, the epidermal proliferative compartment is not composed of equipotent cells, but rather is organized in a functionally hierarchical manner with the most potent quiescent SCs at its apex (i.e., α?6briCD71(dim) ) followed by cycling progenitors (i.e., α?6briCD71(bri) ) and finally early differentiating keratinocytes (i.e., α?6dim).     

Expansion of LTC-ICs and maintenance of p21 and BCL-2 expression in cord blood CD34(+)/CD38(-) early progenitors cultured over human MSCs as a feeder layer

Allogeneic transplantation with umbilical cord blood (UCB) is limited in adult recipients by a low CD34(+) cell dose. Clinical trials incorporating cytokine-based UCB in vitro expansion have not demonstrated significant shortening of hematologic recovery despite substantial increases in CD34(+) cell dose, suggesting loss of stem cell function. To sustain stem cell function during cytokine-based in vitro expansion, a feeder layer of human mesenchymal stem cells (MSCs) was incorporated in an attempt to mimic the stem cell niche in the marrow microenvironment. UCB expansion on MSCs resulted in a 7.7-fold increase in total LTC-IC output and a 3.8-fold increase of total early CD34(+) progenitors (CD38(-)/HLA-DR(-)). Importantly, early CD34(+)/CD38(-)/HLA-DR(-) progenitors from cultures expanded on MSCs demonstrated higher cytoplasmic expression of the cell-cycle inhibitor, p21(cip1/waf1), and the antiapoptotic protein, BCL-2, compared with UCB expanded in cytokines alone, suggesting improved maintenance of stem cell function in the presence of MSCs. Moreover, the presence of MSCs did not elicit UCB lymphocyte activation. Taken together, these results strongly suggest that the addition of MSCs as a feeder layer provides improved conditions for expansion of early UCB CD34(+)/CD38(-)/HLA-DR(-) hematopoietic progenitors and may serve to inhibit their differentiation and rates of apoptosis during short-term in vitro expansion.     

The HOXB4 homeoprotein differentially promotes ex vivo expansion of early human lymphoid progenitors

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.     

Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion

In adults, bone marrow-derived cells (BMDC) can contribute to the structure of various non-haematopoietic tissues, including skin. However, the physiological importance of these cells is unclear. This study establishes that bone marrow-derived epidermal cells are proliferative and, moreover, demonstrates for the first time that BMDC can localize to a known stem cell niche: the CD34-positive bulge region of mouse hair follicles. In addition, engraftment of bone marrow cells into the epidermis is significantly increased in wounded skin, bone marrow-derived keratinocytes can form colonies in the regenerating epidermis in vivo, and the colony-forming capacity of these cells can be recapitulated in vitro. In some tissues this apparent plasticity is attributed to differentiation, and in others to cell fusion. Evidence is also provided that bone marrow cells form epidermal keratinocytes without undergoing cell fusion. These data suggest a functional role for bone marrow cells in epidermal regeneration, entering known epidermal stem cell niches without heterokaryon formation.     

An improved method for culture of epidermal keratinocytes from newborn mouse skin

Reproducible isolation and long term culture of epidermal keratinocytes from transgenic mouse lines is critically needed but most techniques have been unsuccessful. In this report we describe in detail a simplified method to isolate putative keratinocyte stem cells from newborn mouse skin and to maintain them for long term in culture. The cell cultures were established by enzymatically separating keratinocytes from newborn mouse skin. For selecting the putative keratinocyte stem cells for culture, the cells are allowed to attach for 10 minutes on a composite matrix made of type I collagen and fibronectin. Unattached cells were discarded and the attached cells were cultured in a defined culture medium containing low Ca²⁺ concentration, 9% FBS, conditioned medium from newborn mouse skin fibroblasts, and EGF. For subculturing, the cells were seeded on tissue culture plastic. The isolated cells showed the typical basal keratinocyte morphology and expressed the epithelial cell specific integrin v6. The expression level of v6 integrin was comparable to human skin keratinocytes. The keratinocytes were also able to differentiate to form an epidermis in an organotypic culture model. By using the described protocol, the keratinocytes from frozen stocks have been subcultured up to 26 times without change in cell viability, proliferation rate or morphology.