Chapter 78 Flow cytometric analyses and sorting of neural cells for transplantation

D.M Ga\h and J.R. Sladek. J r . (Ed\.) Progress in Braarn Research, Vol. 78 c

19R8 Flxvier Science Publishers

B.V. (Biomedical Divirion)

605 CHAPTER 78

Flow cytometric analyses and sorting of neural cells for transplantation Mary F.D. Nottera, Don M. Gash" and James F. Learyb Department of Neurobiology and Anatomy and Department of Pathology and Laboratory Medicine, University of Rochester School of Medicine and Dentistry, Rochester, N Y 14642, U.S.A.

Introduction Neural implantation, historically employed by developmental neurobiologists to examine regeneration and development within the central nervous system (CNS) has been used successfully to repair neurological disorders in animal model systems (Gash, 1984). The majority of studies has involved fetal CNS tissues which appear to have discrete developmental stages for optimal transplantation and integration with the host (Olson et al., 1984). When peripheral tissue such as adrenal medulla or clonal cell lines such as neuroblastomas serve as donor tissue, survival does not depend on age as much as on the phenotypic plasticity and response to growth factors for integration (Stromberg et al., 1985; Gash et al., 1986). Both phenomena, plasticity and integration, may be reliant, in good measure, upon properties at the cell surface membrane. This structure orchestrates cytodifferentiation, migration and synapse formation during development by responding t o environmental changes via expression of surface glycoconjugates involved in neural self-organization (Raedler and Raedler, 1986). Therefore, the cell membrane may play a pivotal role in regulating the events of recognition, integration and restoration of function in a transplant-host relationship. One powerful method which can be employed to examine the living cell surface is flow cytometry coupled with fluorescent membrane probes. Flow cytometry is a sophisticated, quantitative technique with many applications in biomedical research and clinical medicine (Melamed et al., 1979).

Although largely unexploited by the neurobiologist, this new technology can be applied to examine large numbers of individual cells, allowing rapid measurement of as many as eight different cellular parameters per cell. Some properties which can be measured include cell size, light scatter, two or more colors of fluorescence, spectral distribution of compounds and cell cycle kinetics, all of which offer the advantage for identifying, quantifying and recovering subpopulations of live neurons. In this paper we present data from flow cytometric analyses of several neural cell lines which are being used as alternate sources of donor tissue for transplantation as well as analyses of the developing rat hypothalamus. Also preliminary results of electronically sorted neurons for transplantation and the requirements for obtaining these cells in an optimal condition are discussed.

Principles of flow cytornetry The potential for analysis and sorting of neurons by flow cytometry is limited only by the availability of specific probes and the acquisition of a good single cell preparation. Fig. 1 shows the basic scheme of most flow cytometers. Cell suspensions obtained by enzymatic or mechanical dissociation are transported under pressure in a laminar flow such that cells flow at a high speed in a single file. Cells traverse a sensing region where they are illuminated by one or two lasers and where optical signals are generated and converted to electrical signals. These signals are measured, digitized and collected into discrete bins or channels. Fluores-

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Cytometric analyses of neuronal cell lines

SINGLE CELL SALINE SHEATH

FLOW CHAMBER

TERMINAL

PURIFIED CELL S U B P W U U ~

A

-

0

PURIFIED CELL SUBPOPUUTION

-0

jo

W

lC

UNDESIRED CELLS

Fig. 1. Scheme of a flow cytometer-cell sorter. Cells in suspension flow under pressure one at a time through a laser beam. Scattered light and fluorescence from each cell is sensed, processed and stored. Cells may be sorted based on the intensity of the various parameters being analyzed. Ultrasonic energy is used to break the flow stream after signal detection into small droplets which are charged and electrostatically deflected if they contain the cells of interest.

cently labeled antibodies to specific antigens or fluorescently labeled compounds with affinity for specific compounds such as carbohydrates or DNA are applied to living cells which are excited by the appropriate wavelength of laser light. Fluorescence intensity, fluorescence polarization, light scatter, and time-of-flight-pulse width properties (used for cell sizing) are collected and stored on a cell by cell basis. When cells are to be sorted, the stream of cells is vibrated with sonic energy to obtain droplets and, following the accurate timing from cell analysis to droplet breakoff, the desired droplets containing cells of interest are electronically charged and deflected into cell containers. Cells can be sorted en masse or 'cloned' by single cell sorting into a small holder containing buffer or culture medium, or into 96-well dishes, respectively.

Neural clones have been used extensively as model systems of development, since they have specific neurotransmitters, synapse-forming properties (Bottenstein, 1981), and the facility to differentiate morphologically and biochemically into cells which have many properties of normal neurons (Notter and Leary, 1986). We have employed flow cytometry and fluorescent probes to obtain differentiation profiles of the cell surface glycoconjugates of mouse and human neuroblastomas. Using fluoresceinated lectins with distinct specificities for carbohydrates, we found an increase in Nacetylglucosamine on living N,AB- 1 mouse neural cells following chemically induced differentiation (Notter and Leary, 1987). More specifically, we have studied the presence of a glycoprotein, band 3 which is an integral membrane component found to change with age (Kay et al., 1983) and importantly, to be a substrate for Calpain. Calpain is an enzyme which, along with brain spectrin and band 3 protein, may be involved in modifications necessary for long-term synaptic changes (Siman et al., 1987). N,AB-1 cells were treated with prostaglandin El (10 pg/ml) and dibutyryl CAMP (500 pg/ml) for four days. Fig. 2A,B reveal band 3 protein immunofluorescence on living cell bodies and along neurites. There was an increased concentration of this antigen on differentiated cells which was more dramatic when differentiated human IMR-32 cells were examined for this protein. Human cells differentiated in a similar fashion showed a thirty-fold increase in band 3 expression (Fig. 2C) when compared to mitotic cells on a cell by cell basis measured by flow cytometry. Living neuroblastomas have also been examined for the presence of neural gangliosides which can be studied specifically using tetanus toxin (TT) as a marker (Koulakoff et al., 1982). Ganglioside treatment of N,AB-1 cells induces neuritic sprouting. Gangliosides are important features of the neural cell surface as they serve as specific receptors, and are involved in synaptogenesis (Obato et al., 1977), regeneration (Gorio et al., 1980) and aging (Hitzemann and Harris, 1984). Differentiated N,AB-l cells, which were primarily in G G of the cell cycle, had more toxin-binding 0. 1 gangliosides per cell than mitotic cells (Notter and

607

IMitotic

0Differentiated

Log i n t e g r a t e d g r e e n f l u o r e s c e n c e

Fig. 2. Band 3 glycoprotein detection on the surface of neuroblastoma cells. A. Mitotic mouse N,AB-1 cells stained immunofluorescently for band 3 . B. Differentiated N,AB-1 cells show band 3 staining o n the cell body and along neurites. C. Histograms representing the amount of band 3 protein o n mitotic and differentiated human neuroblastoma cells. Differentiated IMR-32 cells have 30-times more cell surface band 3 protein than mitotic cells as determined by flow cytometric analysis of immunofluorescently stained protein on a single cell basis.

Leary, 1986), while ganglioside treatment of these cells indicated that increased toxin binding was due to specific expression of these glycoconjugates (Notter and Leary, 1985). Furthermore we examined the effect of chemically induced differentiation on the membrane viscosity of neuroblastomas by treatment in suspension with several fluorescent membrane conformation dyes. Anilinonaphthalene sulfonate (ANS) partitions preferentially on the cytoplasmic face of the cell membrane (Dyckman and Welterman, 1970), diphenylhexatriene (DPH) intercalates within the lipid bilayer (Van Blitterswijk et al., 1981) and trimethylamino-DPH (TMA-DPH) interacts with the outer membrane surface (Prendergast et al., 1981). The fluorescence polarization (or anisotropy) of each dye in individual cells was quantified with a flow cytometer by measuring the fluorescence intensities polarized parallel and perpendicular to the excitation beam (a measure of the degree of movement of the dye molecules within the membrane, hence their microenvironment). With all three dyes it was shown that differentiation of N2AB-l cells was followed by an increase in polarization, indicating an increase in mem-

brane rigidity at all levels of the cell membrane (Table I). To establish whether gangliosides play a role in this increased membrane viscosity, mitotic N2AB-1 cells were treated for 24 hours with gangliosides and examined with the same dyes for fluorescence polarization measurements. As seen in Table I ganglioside treatment alone accounted for a large increase in polarization measurements and membrane viscosity. Analyses of developing CNS neurons by flow cytometry

Very few studies have involved the living, developing neuron and flow cytometry to obtain a precisely localized surface measurement of glycoconjugates. Sack et al. (1983) employed fluorescently labeled lectins to reveal specific cell types of the developing mouse cerebellum and quantified the number of cells and the intensity of lectin binding per cell. Cholera toxin was used as a marker of GM, ganglioside with flow cytometry to follow ganglioside expression during proliferation and differentiation of chick retinal cells (Rathjen and Gierer, 1981). Developing rat oligodendrocytes

608

TABLE I Changes in membrane fluidity following differentiation N,AB-1 cells

ANS (100 pM)

DPH (1 pM)

TMA-DPH (1 pM)

Mitotic PGE,/cAMP-treatedb Ganglioside treated'

0.2207 f 0.0015a 0.2275 t 0.0016 0.2461 f 0.0017

0.2170 f 0.0015 0.2236 T 0.0016 0.2692 & 0.0019

0.2560 t 0.0019 0.2647 t 0.0018 0.2923 t 0.0020

Emission anisotropy as a measure of fluorescence polarization of dye bound to individual cells. This quantity represents the variation (t S.E. of the mean) in the mean value of the anisotropy. Living cells were exposed to dye for 30 min at 37°C. Cells were treated with prostaglandin E, (PGE,) (10 &ml) and CAMP (500 pg/ml) for 48 h before being placed in suspension for dye treatment. Cells were treated with gangliosides (200 pg/ml) for 24 h before fluidity measurements.

a

from precursor cells have been traced using monoclonal antibodies and cell sorting (Abney et al., 1983) while Moskal and Schaffner (1986) employed a specific monoclonal antibody to examine expression on embryonic and postnatal hippocampal cells. To extend and expand upon these studies, we have begun to characterize the developmental changes in surface glycoproteins and gangliosides of anterior hypothalamic cells from 19 days post coitus (dpc) embryos as compared with anterior hypothalamic cells from five day neonates. Single cells were treated with fluoresceinated wheat germ agglutinin (FL-WGA), Ulex europaeus (FL-UEA), or with a combined lectin-hapten sugar for blocking experiments. Binding of UEA and WGA was specific as staining was abolished with 200 mM hapten sugars. WGA binding to hypothalamic cells increased with age, while UEA binding was dramatically reduced between 19 dpc and five days postnatal. These findings reflect a possible masking or loss of fucoserich carbohydrates with development of hypothalamic cells. When similar suspensions from fetal and newborn animals were treated with TT and examined for antibody binding by flow cytometry, fetal hypothalamic neurons bound more TT than cells from older animals (mean fluorescence 3.69 vs. 2.13, respectively), indicating more gangliosides on the surface of fetal neurons. However, reanalysis of these data revealed heterogeneity of binding with the presence of subpopulations of low and high TT binding cells in hypothalamic preparations from both ages (Fig. 3). There was a shift in the number of cells in the low binding population seen from fetal day 19 to 5 days postnatal in the anterior hypothalamus; twice

as many older hypothalamic neurons bound TT than fetal neurons when the entire population was analyzed.

Sorting viable neurons for transplantation Several studies reporting the isolation of neurons by cell sorting have employed specific antibody markers for obtaining specific cell types. Abney et al. (1983) used antibodies to TT and GQ1

cR

z Dimly fluorescent subpopulation 8 :Brightly fluorescent subpopulation

A

I

!I

15 day post-cottol Relative mean fluorescence population A = O 1430 Relative mean fluorescence population 0 : 3 25

I

I 5 day newborn

1

'

Relative mean fluorescence population A :0 1533 Relative mean fluorescence population B z 2 00

Fig. 3. Histograms of tetanus toxin (TT) binding as determined by immunofluorescence intensity (x-axis) of hypothalamic neurons. Two populations of low (A) and high (B) TT-binding cells are revealed in both 19 day embryos and 5 day neonates. Twice as many neurons (y-axis) from neonates bind toxin as compared to embryonic hypothalamic neurons, although the amount of binding per cell is greater in the embryonic cells.

609

ganglioside (A,B, monoclonal antibody) to sort total rat brain neurons, while hippocampal cells from 20 dpc embryos were sorted via a specific antibody and cultured for at least one week (Moskal and Schaffner, 1986). However, many antibodies such as the A,B, monoclonal are of the IgM class and have proven to be cytotoxic t o the cells in the presence of complement (Abney et al., 1983). Also, Moskal and Schaffner (1986) reported a large dead cell component to their monoclonal antibody-sorted cell population along with a poor plating efficiency on collagen or poly L-lysine surfaces following sorting of these cells. We have found similar effects with sorted TT, anti-TT labeled hypothalamic neurons from 19 dpc rat embryos (unpublished data). Therefore, sorting cells with specifically bound antibodies may prove to be a problem for obtaining viable cells for transplantation. In this regard, unlabeled cells have been sorted from adult bovine brain and newborn rat brain solely on the basis of light scatter properties which are related to size and shape of cells (Meyer et al., 1980). Using acridine orange for labeling of live cells and light scatter properties, St. John et al. (1986) sorted embryonic mouse spinal cord neurons and glia which were viable in culture for at least five weeks, indicating no detectable damage to these cells per se from the sorter passage. We have sorted fetal anterior hypothalamic cells based on cell size and viability and transplanted these cells into the posterior hypothalamus of neurohypophysectomized adult rats (Lopez-Lozano et al., 1987). Sorted cells survived, migrated in vivo and were positive for neurophysin staining. Therefore, at least some neurons are viable following exposure to laser and hydrodynamic sheer forces used to sort. However, since cell size is not a useful criterion to obtain precise subpopulations of neurons, transplanted cells in this experiment represented many cell types. Therefore, to obtain viable, discrete populations of cells without the potentially harmful effects of antibodies, we adapted a fluorescence retrograde transport technique to label hypothalamic cells for sorting (Rohrer et al., 1983). Fluorescence retrograde labeling has been employed to sort viable motor neurons from rodent (Eagleson and Bennett, 1983) and avian (Calof and Reichart, 1984) sources. Us-

ing a parapharyngeal, infrahyoid, transphenoidal approach, the neural lobes of 20-day postnatal rats were exposed and injected with FL-WGA (2070). Time-course studies revealed that FL-WGA was retrogradely transported to magnocellular neurons of the hypothalamic neurosecretory system within ten hours and remained brightly fluorescent for 48 hours in vivo without loss of fluorescence until four days postinjection. Also, labeled neurons in vivo were unaffected by the tracer, since physiological determination of water consumption and urine osmolalities of these animals remained normal. After a 24 hour period animals were sacrificed, supraoptic and paraventricular nuclei were dissected and dissociated into single cells as described (Notter et al., 1984). Propidium iodide (PI) was added to measure dead cells and the suspension was analyzed and sorted with an EPICS V Sorter based on light scatter and fluorescence intensity. Two-parameter analysis of the labeled hypothalamic preparation was plotted on a contour plot (Fig. 4A). Analysis determined

Green fluorescence

Fig. 4.A. Contour plot of hypothalamic neurons analyzed cytometrically by fluorescence and light scatter. Each contour line represents a different subpopulation of living cells. Those points within the box represent hypothalamic (magnocellular) neurons retrogradely labeled with fluorescent WGA analyzed and sorted by size and fluorescence intensity. Approximately one in 600 cells from the hypothalamic cell preparation was labeled. B. Sorted magnocellular neuron stained immunocytochemically for neurophysin.

610

that one in 611 cells was labeled which, based on the approximation of neurophysin-containing neurons in the hypothalamus, accounts for an 84% recovery rate possible through cell sorting. Flow cytometric analysis of PI-labeled cells indicated that cells had viabilities consistently over 90%, supporting the pre-sort trypan blue dye exclusion results. When sorted cells were stained immunocytochemically for neurophysin, positively stained neurons were detected (Fig. 4B). Therefore, relatively rare populations of neurons can be labeled and sorted.

Concluding remarks Flow cytometry has the potential to provide important analytical data on surface properties of transplantable cells. Surface glycoconjugates such as specific glycoproteins and membrane fluidity measurements of differentiated cells have been quantified for several clonal neural cell lines. Also, levels of specific surface glycoproteins and gangliosides of anterior hypothalamic neurons have been shown to change during development. Since the cell surface is instrumental in recognition and integration in the CNS, cell surface characteristics may dictate survival of transplanted cells and therefore elaboration of these properties is critical. To obtain relatively rare populations of viable neurons for transplantation, retrograde transport of fluorescent markers to discrete CNS neurons followed by isolation by cell sorting is an experimental method which has promise and is being currently employed in transplantation studies.

Acknowledgements We are grateful for the assistance of Drs. Daniel Rohrer and Juan J. Lopez-Lozano in obtaining these data. This work was supported by NIH Grant NS 19711.

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