Ludovic Zimmerlin

Stromal vascular progenitors in adult human adipose tissue

The in vivo progenitor of culture‐expanded mesenchymal‐like adipose‐derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel‐associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture, and tested for adipogenesis. The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/α‐SMA+/CD90±/CD34−/CD31−; the outer adventitial stromal ring (designated supra adventitial‐adipose stromal cells, SA‐ASC) was CD146−/α‐SMA−/CD90+/CD34+/CD31−. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), whereas small vessel endothelium was CD31+/CD34−/CD90− (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31− pericyte subset that may be transitional between pericytes and SA‐ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA‐ASC. Endothelial populations had significantly reduced adipogenic potential compared with unsorted expanded SVF cells. In adipose tissue, perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34− pericytes, and the outermost of CD146−/CD34+ SA‐ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA‐ASC.

Adipogenic Potential of Adipose Stem Cell Subpopulations

Background: Adipose stem cells represent a heterogenous population. Understanding the functional characteristics of subpopulations will be useful in developing adipose stem cell–based therapies for regenerative medicine applications. The aim of this study was to define distinct populations within the stromal vascular fraction based on surface marker expression, and to evaluate the ability of each cell type to differentiate to mature adipocytes. Methods: Subcutaneous whole adipose tissue was obtained by abdominoplasty from human patients. The stromal vascular fraction was separated and four cell populations were isolated by flow cytometry and studied. Candidate perivascular cells (pericytes) were defined as CD146+/CD31–/CD34–. Two CD31+ endothelial populations were detected and differentiated by CD34 expression. These were tentatively designated as mature endothelial (CD 31+/CD34–), and immature endothelial (CD31+/CD34+). Both endothelial populations were heterogeneous with respect to CD146. The CD31–/CD34+ fraction (preadipocyte candidate) was also CD90+ but lacked CD146 expression. Results: Proliferation was greatest in the CD31–/CD34+ group and slowest in the CD146+ group. Expression of adipogenic genes, peroxisome proliferator-activated receptor-&ggr;, and fatty acid binding protein 4, were significantly higher in the CD31–/CD34+ group compared with all other populations after in vitro adipogenic differentiation. This group also demonstrated the highest proportion of AdipoRed lipid staining. Conclusions: The authors have isolated four distinct stromal populations from human adult adipose tissue and characterized their adipogenic potential. Of these four populations, the CD31/CD34+ group is the most prevalent and has the greatest potential for adipogenic differentiation. This cell type appears to hold the most promise for adipose tissue engineering.

Placental Perivascular Cells for Human Muscle Regeneration

Perivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin. In vitro-cultured placental villi released rapidly adhering and migratory CD146+CD34⁻CD45⁻CD56⁻ cells of putative perivascular origin that expressed mesenchymal stem cell markers. CD146+CD34⁻CD45⁻CD56⁻ perivascular cells isolated and purified from the placental villi by flow cytometry were indeed highly myogenic in culture, and generated dystrophin-positive myofibers, and they promoted angiogenesis after transplantation into SCID/mdx mouse muscles. These observations confirm the existence of mesenchymal progenitor cells within the walls of human blood vessels, and suggest that the richly vascularized human placenta is an abundant source of perivascular myogenic cells able to migrate within dystrophic muscle and regenerate myofibers.

Rare Event Detection and Analysis in Flow Cytometry: Bone Marrow Mesenchymal Stem Cells, Breast Cancer Stem/Progenitor Cells in Malignant Effusions, and Pericytes in Disaggregated Adipose Tissue

One of the major strengths of Flow Cytometry is its ability to perform multiple measurements on single cells within a heterogeneous mixture. When the populations of interest are relatively rare, analytical methodology that is adequate for more prevalent populations is often overcome by sources of artifacts that become apparent only when large numbers of cells are acquired. This chapter presents three practical examples of rare event problems and gives detailed instructions for preparation of single cell suspensions from bone marrow, malignant effusions, and solid tissue. These examples include detection of mesenchymal stem cells in bone marrow, characterization of cycling/aneuploid cells in a breast cancer pleural effusion, and detection and subset analysis on adipose-derived pericytes. Standardization of the flow cytometer to decrease measurement variability and the use of integrally stained and immunoglobulin capture beads as spectral compensation standards are detailed. The chapter frames rare event detection as a signal-to-noise problem and provides practical methods to determine the lower limit of detection and the appropriate number of cells to acquire. Detailed staining protocols for implementation of the examples on a three-laser cytometer are provided, including methods for intracellular staining and the use of DAPI to quantify DNA content and identify events with ≥2N DNA. Finally, detailed data analysis is performed for all three examples with emphasis on a three step procedure: (1) Removal of sources of interference; (2) Identification of populations of interest using hierarchical classifier parameters; and (3) Measurement of outcomes on classifier populations.

Localization of CD44 and CD90 Positive Cells to the Invasive Front of Breast Tumors

A variety of markers have been proposed to identify breast cancer stem cells. Here, we used immunohistostaining and flow cytometry to analyze their interrelationships and to sort cells for tumorigenicity studies.

Human adipose stromal vascular cell delivery in a fibrin spray

BACKGROUND AIMS Adipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system. METHODS SVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography. RESULTS SVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells. CONCLUSIONS Human adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

BACKGROUND AIMS Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs. METHODS We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4-T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler. RESULTS The majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10(10) total cells, 6.2 ± 2.2 × 10(8) of which were CD45(+) CD34(+). Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34(+) CD90(+) CD117(dim) hematopoietic stem cells (15.5 ± 7.5% of the CD34 (+) cells) and CD45(-) CD73(+) CD105(+) mesenchymal stromal cells (0.04 ± 0.04% of the total cells). CONCLUSIONS This procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.

Pericytes: A Universal Adult Tissue Stem Cell?

In this issue of Cytometry part A, Montiel-Eulefi et al. describe a neurogenic cell population residing in the vascular wall of rat aortas. Although the authors did not characterize them in situ, these aorta-derived cells were shown to express well-known pericytic markers such as CD90, α-smooth muscle actin, and the platelet derived growth factor receptors α and β (1,2) after a short period in culture. The authors also report the native expression of the neural stem cell marker nestin (3), which in combination with the chondroitin sulfate proteoglycan NG2 marks both pericytes and neurogenic progenitors (1,3). They validated the neurogenic potential of cultured aorta-derived pericytes by showing the acquisition of mature neural marker expression and functional neural-like stimulus-response. In this study, the pluripotency associated marker stage-specific embryonic antigen 1 (SSEA-1) was detected on aorta-derived cultured pericytes only after induction of neuronal differentiation, in contrast to SSEA-1 expression by human microvascular pericytes, which has been reported to be constitutive (4). A variety of multilineage stem/progenitor cells have been described in the recent literature, most of them being identified retrospectively following primary culture. These include multipotent adult progenitor cells (5), mesenchymal stem cells (MSC) (6) and adipose-derived stem cells (ASC; Ref. 7). Most of these multipotent cell populations have been localized in vivo to a niche in the microvasculature wall, including MSC (8) and ASC (4,7), although MSC have been also isolated from larger vessels, both arteries (9) and veins (10). At the clonal level, the multilineage potential of MSC (and other comparable stem cell populations) has been demonstrated in vitro for mesenchymal lineages (adipogenic, osteogenic, chondrogenic, and either skeletal or smooth muscle potential; reviewed in Ref. 11). Further, trans-differentiation across germ layers has been reported by various groups, including differentiation toward neuronal lineages (reviewed in Ref. 11). During embryogenesis, MSC originate in two distinct waves, the first from the neural crest. A second wave appears to derive from the mesoderm (12). The relationship between MSC and pericytes in the adult is not immediately clear. In a variety of tissues, pericytes have been proposed to be the immediate progenitors of MSC (8). Brain microvascular pericytes have been previously shown to differentiate into multiple neuronal lineages (3), consistent with the observation that pericytes residing in the face and forebrain originate, like neurons, from the neural crest (13). In this work, the authors successfully differentiated aorta-derived pericytes toward the neurogenic lineage, suggesting that pericytes originating outside the neural crest can also be induced to differentiate down the neuronal lineage pathway. The constitutive coexpression of nestin and NG2 in tissue pericytes is consistent with neuronal-lineage priming, similar to that observed in mesenchymal lineages (6), and potentially explaining the cross-germ layer plasticity reported in the literature (11). Our own data, relating adipose pericytes, which associate with the microvasculature (7), to the mesenchymal-like supra-adventitial adipose stromal cells (SA-ASC), which surround small vessels in an annular fashion, support the interpretation that pericytes are the progenitors of MSC in the adult. Figure 1A shows a hypothetical differentiation scheme based on multiparameter flow cytometric analysis of the stromal vascular fraction of disaggregated adipose tissue. According to this scheme, CD146+, CD90±, CD34− pericytes differentiate from the inside out, giving rise to MSC-like CD146−, CD90+, CD34+ SA-ASC through a rare CD146+, CD90+, CD34+ transit amplifying cell of high proliferative capacity. The SA-ASC undergo spontaneous adipogenesis after reaching confluence in culture and presumably give rise to mature adi-pocytes in vivo. In keeping with the findings of Montiel-Eulefi et al. human adipose stromal vascular cells also occasionally give rise to CD146+ cells with neuronal morphology after two weeks in culture in the presence of EGM2 (Fig. 1B). CD 146 is not just a pericyte marker. In the neurobiology literature it is known as gicerin, a ligand for nerve outgrowth factor with an important role in neurite extension and synaptogenesis (14). Figure 1 (A) Differentiation scheme for pericytes, transit amplifying cells and supra-adventitial stem cells in adipose tissue. Flow cytometry was performed on 10 freshly isolated human adipose stromal vascular preparations according to previously published methods. ... Taken together, these data may help answer the conundrum posed by adult tissue stem cells: Does each tissue have its own oligopotent stem cell which arises during embryogenesis after lineage specification, or is there a universal pluripotent adult tissue stem cell which has escaped lineage restriction (15)? If the findings of Montiel-Eulefi et al., which even large vessels harbor pericytes capable of cross germ-layer differentiation, prove to be generalizable, then pericytes may represent something approaching the universal adult tissue stem cell, and the adventitia of large and small vessels may be their niche.

KIT (CD117) Expression in a Subset of Non-Small Cell Lung Carcinoma (NSCLC) Patients

We have previously described the expression of CD44, CD90, CD117 and CD133 in NSCLC tumors, adjacent normal lung, and malignant pleural effusions (MPE). Here we describe the unique subset of tumors expressing CD117 (KIT), a potential therapeutic target. Tumor and adjacent tissue were collected from 58 patients. Six MPE were obtained before therapy. Tissue was paraffin embedded for immunofluorescent microscopy, disaggregated and stained for flow cytometry or cryopreserved for later culture. The effect of imatinib on CD117high/KIT+ tumors was determined on first passage cells; absolute cell counts and flow cytometry were readouts for drug sensitivity of cell subsets. Primary tumors divided into KITneg and KIT+ by immunofluorescence. By more sensitive flow cytometric analysis, CD117+ cytokeratin+ cells were detected in all tissues (1.1% of cytokeratin+ cells in normal lung, 1.29% in KIT “negative” tumors, 40.7% in KIT+ tumors, and 0.4% in MPE). In KIT+/CD117high, but not KIT+/CD117low tumors, CD117 was overexpressed 3.1-fold compared to normal lung. Primary cultures of CD117high tumors were sensitive to imatinib (5 µM) in short term culture. We conclude that NSCLC tumors divide into CD117low and CD117high. Overexpression of CD117 in CD117high NSCLC supports exploring KIT as a therapeutic target in this subset of patients.

Capturing Human Naïve Pluripotency in the Embryo and in the Dish

Although human embryonic stem cells (hESCs) were first derived almost 20 years ago, it was only recently acknowledged that they share closer molecular and functional identity to postimplantation lineage-primed murine epiblast stem cells than to naïve preimplantation inner cell mass-derived mouse ESCs (mESCs). A myriad of transcriptional, epigenetic, biochemical, and metabolic attributes have now been described that distinguish naïve and primed pluripotent states in both rodents and humans. Conventional hESCs and human induced pluripotent stem cells (hiPSCs) appear to lack many of the defining hallmarks of naïve mESCs. These include important features of the naïve ground state murine epiblast, such as an open epigenetic architecture, reduced lineage-primed gene expression, and chimera and germline competence following injection into a recipient blastocyst-stage embryo. Several transgenic and chemical methods were recently reported that appear to revert conventional human PSCs to mESC-like ground states. However, it remains unclear if subtle deviations in global transcription, cell signaling dependencies, and extent of epigenetic/metabolic shifts in these various human naïve-reverted pluripotent states represent true functional differences or alternatively the existence of distinct human pluripotent states along a spectrum. In this study, we review the current understanding and developmental features of various human pluripotency-associated phenotypes and discuss potential biological mechanisms that may support stable maintenance of an authentic epiblast-like ground state of human pluripotency.