Identifying and tracking neural stem cells

Available online at www.sciencedirect.com R

Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

www.elsevier.com/locate/ybcmd

Identifying and tracking neural stem cells Jingli Cai,a,b,1 Tobi L. Limke,b,1 Irene Ginis,b and Mahendra S. Raoa,b,* b

a Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84132, USA Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, Gerentology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA

Submitted 21 April 2003 (Communicated by M. Lichtman, M.D., 4/25/03)

Abstract Hematopoietic stem cells, unlike neural stem cells, can be readily identified and isolated from developing and adult cell populations using positive and negative selection criteria. Isolating stem cells and progenitor cells from neural tissue has been more difficult because of difficulties in separating cells in solid tissue, the limited numbers of stem cells that persist in the adult, and the paucity of rigorously characterized markers. Nevertheless, strategies that have worked successfully in hematopoietic stem cell isolation can be adapted to isolate multiple classes of stem and progenitor cells from neural tissue. Neural stem cells also share cellular and molecular properties with other stem cell populations that may serve as surrogate identifiers of multipotentiality. Such potential markers are described. Unlike hematopoietic stem cells, tracking neural cells after transplantation is both necessary and more difficult. It will therefore be necessary to develop invasive and non-invasive strategies to follow transplanted cells and develop useful quantifiable readouts. Some potential strategies are described and current results are discussed. © 2003 Elsevier Science (USA). All rights reserved.

Introduction The term “stem cell” is generally used to define a cell that is multipotent and has a theoretically unlimited capacity to maintain self-renewal and differentiate into functional, mature cells. Stem cells can undergo symmetric divisions to increase the population of stem cells or asymmetric divisions to produce another stem cell and a blast or intermediate precursor/progenitor cell which is more restricted in its differentiation capacity or a symmetric terminal differentiation to generate two differentiated daughter cells. Stem cells are classified as pluripotent, such that the stem cell can give rise to cells of all three germ layers (endoderm, ectoderm, and mesoderm), or multipotent, with the stem cell giving rise to a cells in a specific tissue lineage (neural, hepatic, etc.). Pluripotent stem cells are typified by embryonic stem (ES) cells, which are derived from the inner cell mass of blastocysts. Multipotent stem cells, on the other hand, are derived from various tissues in the fetal and/or * Corresponding author. Fax: ⫹1-410-558-8323. E-mail address: [email protected] (M.S. Rao). 1 These authors contributed equally to the manuscript.

adult organism and include hematopoietic stem cells (HSC), neural stem cells (NSC), hepatic stem cells, etc. While multipotent stem cells from different tissues all exhibit the defining properties of stem cells (multipotency and capacity to self-renew and differentiate), there are critical differences in their biological properties in terms of their cytokine dependence, antigen expression, and functional abilities. The best characterized stem cell is the HSC, which was first described in the late 1940’s– early 1950’s, when it was discovered that injection of blood marrow cells could rescue animals from fatal marrow aplasia caused by high-dose irradiation [1–3]. As a result, stem cells purified from bone marrow and cord blood have been routinely harvested, stored, and used therapeutically to treat a variety of bloodrelated disorders, ranging from cancer to multiple sclerosis and immune deficiency, for the past 40 years. Unfortunately, the success of bone marrow transplantation has not yet been emulated in other fields, which are still in the early stages of experimentation. Fetal tissue transplants have been performed in the central nervous system (CNS) for treatment of Parkinson’s disease with varying degrees of success (see [4] for review), and in the pancreas for treatment of diabetes (see [5] for review). Progenitor cells have also been

1079-9796/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1079-9796(03)00130-X

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

used for autologous cellular replacement of skin and cartilage. However, HSC-based therapy is the only routinely used stem cell therapy in modern medicine. In order for the success of bone marrow transplantation to be translated into success in other fields, it is useful to understand the key features of HSCs and HSC-based therapy. In this review, we address the basic biological properties of HSCs in comparison to another type of stem cell, the NSC, in order to understand similarities and differences between these two types of cells that need to be considered when designing therapeutic strategies using these cells.

Hematopoietic stem cells Biological properties Since the first description of HSCs over half a century ago, the field of HSC biology has developed reliable methodology for the characterization, isolation, and study of HSCs in vitro and in vivo. Given the vast knowledge of HSC biology, it is tempting to use methods used to isolate and purify HSCs to study NSCs; thus, we present a brief review of HSC biology to provide an understanding of the approaches used to identify and isolate NSCs. Bone marrow HSCs are functionally defined by their ability to self-renew and to differentiate into mature blood cells of all types, as indicated by clonal analysis in vitro and retroviral labeling studies in vivo (see [6] and [7] for review). The pool of HSCs has been extensively characterized and consists of several subpopulations distinguishable on the basis of both phenotype and function (Fig. 1A). Long-term reconstituting HSCs (LT-HSC) exhibit the greatest capacity for self-renewal and will give rise to all hematopoietic lineages throughout the life of the organism. LT-HSCs give rise to short-term HSCs (ST-HSC), which also exhibit multipotentiality but will do so for only 8 –10 weeks. ST-HSCs give rise to oligopotent progenitors restricted to either the lymphoid or the myeloid lineage. HSCs have also been identified in the fetal liver (FL); however, these cells differ from bone marrow HSCs both in phenotype and function. Bone marrow stem cells in the adult are primarily of the ST-HSC and lineage-restricted variety, while FL stem cells are primarily thought to be LT-HSCs. LT-HSCs, ST-HSCs, and progenitor cells represent a continuum of differentiation rather than abrupt transitions or irrevocable stages of differentiation (see [8] for review). Because of their long-term self-renewal capacity, the LT-HSC population is the focus of intense study in an attempt to define factors that support long-term self-renewal in vitro and in vivo, thereby allowing for expansion of the stem cell pool and increased numbers of progeny following differentiation. LT-HSCs can be divided into subpopulations which can be prospectively isolated on the basis of expression (or lack of expression) of specific cell-surface epitopes. The most common approach is to use fluorescence-activated cell sorting (FACS) to sepa-

19

rate and purify specific HSC subpopulations for study in isolation. This separation is crucial because, as mentioned above, the HSC pool consists of a heterogeneous pool of cells with self-renewal capacity but distinct cell-surface markers (Table 1). Both LT-HSCs and ST-HSCs in the adult bone marrow are contained within the lineage marker⫺/lo (Lin⫺/lo), c-Kit⫹, Sca-1⫹, Thy1.1lo subset of marrow cells, referred to as KTLS cells. Transplantation of a single KTLS cell into the bone marrow of a lethally irradiated mouse is sufficient to produce long-term, multilineage hematopoietic engraftment in the host animal [9]. The murine HSC pool can be further differentiated by expression of markers such as Flk-2 (which is absent in LT-HSCs and present in STHSCs) [10] and CD34 (which is not expressed or expressed at low levels in LT-HSC and is expressed in ST-HSCs). Multilineage progenitors are also Lin⫺, c-Kit⫹, Sca-1⫹, and Flk-2⫹; however, they have lost expression of Thy-1.1 that is observed in low levels in LT-HSCs and ST-HSCs. Marker expression can also vary by species; for example, murine LT-HSCs express low to no CD34, while human HSCs are highly enriched in the CD34⫹ bone marrow fraction [11]. Using multichannel cell sorting, these populations can be prospectively isolated and studied. Subpopulations within the HSC pool can also be distinguished using other criteria, including size [12], cell cycle status [13], rhodamine-123 staining [14,15], and Hoechst 33342 staining [16]. Rhodamine-123 accumulates into mitochondria on the basis of its highly negative inner mitochondrial membrane potential; thus it can be used as an indirect measure of mitochondrial activity. Bone marrow cells which exhibit low levels of rhodamine fluorescence also have the greatest proliferative capacity, as assessed using colony-forming assays and repopulation capacity, and are considered more quiescent and thus more primitive than high-fluorescence cells [15]. Similarly, Hoescht 33342 can be used to distinguish HSCs from non-HSCs in living cells using FACS. Murine and human HSCs express the multidrug resistance protein (MDR/MDR related) which allows for rapid efflux of the Hoechst dye from HSCs; thus, FACS of cells with low Hoechst staining results in a side population of cells which are Lin⫺ and exhibit long-term selfrenewal capacity and multilineage potential [16]. Combining the strategy of isolating side-population cells with selection based on positive or negative expression of specific markers, one can effectively isolate specific subpopulations of HSCs and multipotential precursors from the bone marrow population.

Neural stem cells Biological properties While the field of NSC biology is much younger than HSC biology, there have been great advances in recent years in understanding neural development and the basic biology

20

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27 Table 1 Positive, negative, and universal selection markers for HSCs

Positive selection markers

Thy1.1lo Negative selection markers Universal selection markers

Marker

Type(s) of HSC

c-Kit⫹ Sca-1⫹ Flk-2⫹ LT-HSC, ST-HSC Lin⫺/lo CD34⫺/lo Hoechst 33342lo Rhodamine-123lo

LT-HSC, ST-HSC LT-HSC, ST-HSC ST-HSC LT-HSC, ST-HSC LT-HSC LT-HSC LT-HSC

of NSCs. NSCs follow a similar pattern of development as HSCs, in that there is a multipotent stem cell which divides to produce more stem cells and lineage-restricted progenitor cells (Fig. 1B). In the developing neural tube, multipotent neuroepithelial cells (NEP) give rise to neuron-restricted precursors (NRP) and glial-restricted precursors (GRP), which then differentiate into mature neurons or glia (oligodendrocytes and astrocytes), respectively. It should be noted that this lineage pathway is a bare outline of the process, with an undetermined number of intermediate precursors possible between different stages of development. For example, astrocyte development may involve an intermediate astrocyte-restricted precursor (AP) which expresses CD44 and may be derived from the GRPs [17], and multiple classes of stem cells may exist (reviewed in [18]). While the exact cellular identity of the stem cell population(s) in the adult brain is still being debated, it is known that multipotent stem cells do exist in discrete locations which can give rise to neurons, astrocytes, and oligodendrocytes. The primary commonality between HSCs and NSCs is the existence of stem cells and more differentiated progenitor cells. However, NSCs differ from HSCs in several critical ways which affect their biology and their potential therapeutic uses (Table 2). The first is the relatively small number and spatial restriction of NSCs present in the adult brain. NSCs are localized to the subventricular zone (SVZ) surrounding the lateral ventricles and the subgranular layer (SGL) of the hippocampus (although there is considerable debate over whether the proliferative cells in the SGL are true stem cells or are lineage-restricted precursor cells; see [19]). Second, the amount of basal cell turnover is low, especially when compared to blood cells. For the most part, cells in the CNS last the lifetime of the individual; thus cell replacement is not the norm for this tissue. Production of most brain cells ceases soon after birth; thus the normal developmental cues promoting cell proliferation and differ-

21

Table 2 Comparison of stem cell characteristics that make HSC therapy successful and may be a problem for NSC replacement HSC

NSC

Most primitive population Appropriate development signals in adult Stem cell niche

Yes Yes

Maybe No

Yes

Homing/delivery of cells Ongoing cell turnover

Yes Yes

Connectivity and integration

Not necessary

Number of cells available Reliability of cell markers for selection

Large Established

Not in most brain regions Difficult Limited in most brain regions Critically necessary Limited Controversial

entiation are generally absent in the adult (unlike the lifelong presence of proliferation signals in the hematopoietic system). Third, NSCs are limited in the types of neurons they can generate: NSCs in the SVZ become interneurons in the olfactory bulb, while NSCs in the SGL become granule neurons within the hippocampus. While there are reports of NSCs being isolated from other brain regions, it is generally accepted that neurogenesis under physiological conditions is not common outside the SVZ and SGL. NSCs also rely on positional information in a way that HSCs do not. Finally, transplantation of HSCs requires the creation of a niche (usually through the removal of endogenous cells using chemotherapy) to give transplanted cells a competitive advantage; this process of “niche creation” has not been tested in the nervous system and in fact is an uncertain process given the restricted nature of the neural stem niche/neurogenic regions. Unlike HSCs, NSCs can be isolated and propagated in culture for extended periods of time, allowing for the creation of NSC “banks” with a large number of cells for study. However, these proliferative populations often contain several types of proliferative cells, including stem cells and progenitor cells; thus one goal of NSC biology is to find markers which positively identify NSCs. Unlike the HSC system, definitive markers for NSCs have remained elusive. Lineage-restricted precursor cells have been identified in the developing spinal cord and can be identified by their expression of PSA-NCAM/E-NCAM (for NRPs) or A2B5 (for GRPs). Different stages of radial glia and oligodendrocyte development can be identified on the basis of a number of epitopes. However, no good markers have emerged for

Fig. 1. Lineage relationship in adult hematopoietic system and early CNS development. (A) In the adult hematopoietic system, long-term HSCs (LT-HSC) divide to produce more LT-HSCs and short-term HSCs (ST-HSC). ST-HSCs give rise to oligopotent progenitor cells which are restricted to the myeloid lineage (CMP) or the lymphoid lineage (CLP). CMPs and CLPs then further divide and differentiate into mature blood cells. (B) In the CNS, multipotent NSCs divide to produce more NSCs and progenitor cells which are classified by the type of mature cell they ultimately produce: neuron-restricted progenitors (NRP) become neurons, while glial-restricted progenitors (GRP) become glia (both oligodendrocytes and astrocytes). An undetermined number of intermediate precursors may also exist; for example, there is evidence suggesting the existence of a GRP-derived astrocyte-restricted progenitor (AP) [17].

22

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

Table 3 Potential markers for NSCs Marker

Comments

Nestin Musashi Hu Nucelostemin Sox-1 Sox-2

Probably expressed in all dividing neural populations Expressed by NSC and progenitor populations Expressed by NSC and progenitor populations Recently described factor; may be specific for dividing populations Appears to be relatively specific for NSCs; expression may transiently persist in some progenitor populations Expression is localized to neurogenic regions in embryo and adult; also expressed in all neurosphere-forming cells Has been used to prospectively identify NSCs Identifies NSCs; expression appears to last beyond stem cell stage Has been used to prospectively identify NSCs Present in all stem cell populations, but may not be restricted to stem cells May identify quiescent stem cell populations, but will not identify rapidly dividing cells; not useful for separating NEP cells from other cells Appears relatively specific for stem cells, including NSCs Appears to identify NSCs in mixed populations of cells Has been used successfully to enrich for stem cell populations at many stages of development

SSEA-1/LeX AC133 (prominin) Response to acetylcholine Telomerase activity/TERT expression Low Hoechst/rhodamine staining ABCG2 expression Aldefluor labeling Absence of differentiation markers

NSCs, other than lack of expression of markers for lineagecommitted cells. Expression of the intermediate filament nestin has been described as defining a NSC; however, nestin is also expressed by radial glia and immature neurons, making this marker imprecise. As discussed below, there other potential NSC markers, including Sox1, Sox2, FGFR4, and SSEA-1; however, their utility is still being explored by several laboratories. While we cannot discuss the entire repertoire of potential NSC markers, a more comprehensive list is provided (Table 3).

Common strategies for prospective isolation of stem cells Because HSCs and NSCs share the properties of multipotentiality and self-renewal, it is not unreasonable to hypothesize that these two types of cells may share common traits (expression of transcription factors, receptors, etc.) which confer these properties and thus may be considered “universal properties” of stem cells. It is also logical to use similar strategies to prospectively isolate stem cells from a mixed population. The primary method for sorting HSCs is FACS; the following sections will focus on the success and failure of applying HSC-based strategies to NSC isolation and will suggest several potential markers which may prove useful in future studies.

Positive selection As described above, HSCs can be prospectively isolated on the basis of their expression of c-Kit, Sca-1, and Thy1.1. Unfortunately, a similar set of markers in the CNS has been more elusive (Table 3). Several promising candidates, such as nestin and musashi, are also expressed in other non-NSCs and thus are not reliable markers for positive selection of NSCs. The issue is further complicated by the fact that several different cell populations behave like NSCs, which may indicate the existence of several sub-populations of NSCs within the CNS (Table 4). Additionally, there are many populations of lineage-restricted progenitors which are not multipotent but may be better suited for transplantation therapy. Transplantation of NSCs into the adult murine spinal cord results in gliogenesis but not neurogenesis; however, transplantation of PSA-NCAM-expressing NRPs resulted in neurogenesis [20]. Thus, the ability to separate NSCs from lineage-restricted progenitors and differentiated cells becomes a powerful tool for developing realistic therapeutic strategies in the CNS. For positive selection, there are several markers that are potentially unique to NSCs and thus may prove useful for NSC isolation. Such markers include the POU transcription factors Sox1 and Sox2 and the plasma membrane proteins FGFR4 and SSEA-1, all of which are highly expressed in

Table 4 Potential markers to identify sub-populations of NSCs Marker

Comments

GFAP S100␤/GLAST MAP2/␤III tubulin RC1/RC2/vimentin PSA-NCAM A2B5

May May May May May May

distinguish between VZ/cortical stem cells and radial glia/astrocyte-derived stem cells distinguish between VZ/cortical stem cells and radial glia/astrocyte-derived stem cells recognize ependymal stem cells identify radial glia-derived stem cells distinguish cortical/parenchymal stem cells from other stem cell populations; may also recognize ependymal stem cells distinguish between glial precursors and other dividing progenitor cell populations

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

the multipotent NSCs and/or pluripotent stem cells [21–23]. Because FGFR4 and SSEA-1 are membrane proteins, development of specific antibodies recognizing their extracellular epitopes might allow NSCs to be separated using positive sorting. However, preliminary results within our laboratory suggest that FGFR4 expression may be limited to embryonic NSCs (personal observation). Sox1 and Sox2 are transcriptional factors; thus direct labeling for live cells is currently impossible. Several groups have generated Sox2– GFP transgenic mice to visualize the expression of Sox2 with GFP expression driven by the Sox2 promoter (L.H. Pevny, personal communication). In these mice, GFP fluorescence correlates very well with expression of Sox2 and is found in areas of high neurogenesis in both the embryo and the adult. Positive selection for GFP⫹ cells results in multipotent populations of cells that give rise to cells of all three lineages in the CNS. By sorting out GFP⫹ cells from these transgenic mice, researchers can easily get a pure population of NSCs for study in isolation. Negative selection Our laboratory and others have had success isolating multipotent NSCs using a negative selection strategy, in which cells not expressing epitopes commonly expressed in lineage-restricted cells are separated using FACS. In addition to the previously described the NRP-specific E-NCAM and the GRP-specific A2B5, other membrane markers for mature neural cell types, such as O4 for oligodendrocytes and CD44 for astrocytes, can also be used to separate NSCs from more mature mixed populations. Depletion of cells expressing E-NCAM and A2B5 from the E14.5 rat spinal cord using FACS results in a multipotent NEP cell population capable of giving rise to neurons, glia, and oligodendrocytes [22]. The purity of this population has been demonstrated by immunostaining and RT-PCR for expression of markers typically found in lineage-restricted and mature cells. This strategy has been applied to the late embryonic rat hippocampus, in which E-NCAM⫺/A2B5⫺ cells have been isolated and grown as bFGF- and EGF-responsive neurospheres (Fig. 2). A similar strategy has been employed by selecting cells which do not express markers of differentiation or apoptosis [24]. Others have isolated NSCs from the periventricular region of the adult rat brain on the basis of nestin expression combined with low expression of PNA binding and HSA proteins in the subventricular zone [25]. In the late embryonic rat hippocampus, CD24 (HSA) is expressed in all regions except the highly proliferative ventricular zone, suggesting that it might be a useful tool for negative selection strategies (personal observation). One good candidate for negative selection is a non-toxic fluorescence-conjugated C-terminal of tetrodotoxin (TTX-C). Most mature neurons express TTX-sensitive sodium channels; thus these cells can be labeled by TTX-C while NSCs are not (unpublished data). Thus, negative selection strategies are also a promising strategy for isolating NSCs; how-

23

Fig. 2. Neurospheres from E-NCAM⫺/A2B5⫺ cells derived from the E18 rat hippocampus and maintained for 6 days in Neurocult medium containing bFGF and EGF. While the ability to form neurospheres does not necessarily indicate that the cells are derived from a pure stem cell population (as GRPs and NRPs are often components of neurospheres), the ability to form neurospheres generally indicates that a stem cell population is present.

ever, its usefulness for isolating NSCs from other brain regions has not yet been determined. Universal stem cell markers Stem cells derived from different tissues have some similar properties, including proliferative quiescence (when not induced to divide and differentiate) and, in some cases, the shared expression of specific proteins. Thus, strategies which separate HSCs from non-HSCs on the basis of these properties offer a rational approach for separating NSCs from non-NSCs. Hoechst 33342 and rhodamine-123 are commonly used to separate HSCs from non-HSCs in the mixed cell population on the basis of low fluorescence of either dye [14,26]. In rat NEP cells, rhodamine-123 proved unsuccessful as a means of separating NSCs from other cells. Rhodamine-123 effectively differentiated between actively dividing cells and non-dividing cells as confirmed by bromodeoxyuridine incorporation [22]. However, rhodamine fluorescence could not differentiate between NSCs and the lineage-restricted NRPs and GRPs [22]. Similar results were obtained using Hoechst dye to separate NEP cells from other cells in the mixed population. Primitive HSCs are isolated using FACS to collect the Hoechstnegative/low side population of cells, which exhibit lower fluorescence as a result of rapid dye efflux through specific transporters; however, this approach has not proven useful for separation of multipotent NEP cells from other precursor cells [22]. Although NEP cells express one of the efflux pumps, ABCG2 (Bcrp1), they are still labeled brighter than differentiating precursor cells by Hoechst dye; however, the difference is not sufficient to allow for separation of this population. Examination of the “side population” from

24

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

Fig. 3. The fluorescent-conjugated substrate Aldefluor labels multipotent NEP cells as a result of their higher ALDH activity, compared to more differentiated cells. (A) Fluorescent image of NEP cells labeled with Aldefluor. (B) Phase contrast of the same field shown in A.

mixed neural cells revealed that the population consisted entirely of cell debris, indicating that the side-population strategy does not work for our NSC isolation. The mechanism underlying the lack of dye efflux from NSCs has not yet been determined. However, we hypothesize that a difference in metabolic activity may underlie this difference. Most HSCs are quiescent cells located in stem cell niches and activated only as necessary. In contrast, NEP cells are actively dividing cells and thus have higher metabolic activity. The difference is not due to lack of expression of ABCG2, as it can be detected by RT-PCR and immunostaining. Interestingly, its expression is down-regulated in lineage-restricted precursors ([22], unpublished data), making its expression, if not its ability to efflux Hoechst dye, a promising marker for NSCs. Experiments are currently underway to determine whether antibodies recognizing the external epitope of ABCG2 can be used to separate NSCs from other cell populations. Telomerase activity appears to correlate with self-renewal in both HSCs and NSCs. Telmoerase is an RNA/ protein complex responsible for lengthening telomeric DNA, a critical factor in determining cellular senescence. Telomerase is expressed in mouse fetal liver and bone marrow HSCs and is down-regulated as HSCs differentiate into multipotent progenitor populations [27]. In the embryonic murine brain, telomerase activity is high during development but is down-regulated by 16 days post-birth and cannot be detected in mature neurons or glia [28]. While it is tempting to use telomerase activity as a marker for stem cells, it is actually an indicator of dividing cells, not necessarily multipotent cells, as telomerase activity and expression of tert (the catalytic subunit of telomerase) cannot distinguish between NEP cells and other dividing cells [22].

More recent experiments suggest that aldehyde dehydrogenase (ALDH) expression and activity level may be a shared characteristic between HSCs and NSCs. ALDH catalyze the pyridine nucleotide-dependent oxidation of aldehydes to acids and has a protective role by providing resistance to alkylating agents [29,30]. Both expression level and enzyme activity of ALDH are high in the HSCs [31,32]. Using fluorescent substrates for ALDH, other investigators have successfully separated HSCs from mixed cell populations [31,33]. Using Aldeflour, a fluorescenceconjugated ALDH substrate, to test the activity of ALDH in NEP cells, we found that NEP cells also had high activity of ALDH, as indicated by bright, specific labeling with Aldefluor (Fig. 3). As cells become more differentiated, Aldefluor labeling intensity is noticeably fainter (personal observation); thus the difference between high and low fluorescence intensity may allow for cell sorting. Based on this observation, we can draw a preliminary conclusion that ALDH activity is universally high in different stem cell populations. This reagent will provide another key to isolate NSCs using a positive-selection FACS methodology. Transplanting and tracking cells in vivo The advantage of purifying bone marrow cells for the HSC population has been demonstrated in transplantation studies using human CD34⫹ cells, in which HSCs are highly enriched [11]. Autologous and allogenic transplantations of CD34⫹ cells result in rapid reconstitution of all blood lineages and a lower incidence of graft-versus-host disease, compared to transplantation of the heterologous bone marrow population or peripheral blood cells [34 –36]. This approach of using the most primitive cell to replace

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

25

Table 5 Methods for monitoring cells after transplantation Labeling cells

Using intrinsic differences between donor and host

Non-invasive monitoring

Fig. 4. General strategy for isolation and expansion of NSCs for therapeutic purposes.

lost or damaged cells has proven highly successful for using HSCs in therapeutic applications. The ability of HSCs to last the lifetime of an individual, combined with the ability to purify the HSC population on the basis of CD34 expression, allows for transplantation of a highly enriched population capable of replacing all cells in the blood lineage. Additionally, it is relatively easy to obtain donor cells (once a donor has been identified) in sufficient numbers to repopulate the host’s hematopoietic system. Given the ability of the KTLS cell to repopulate the entire repertoire of mature blood cells, there is no advantage to using more developed lineage-restricted progenitor cells, thus obviating the need for this additional level of purification. As discussed above, the general strategy underlying HSC-based cell therapy involves isolation and transplantation of the most primitive cell population, which will differentiate into all cell types of the blood lineage. This strategy works because (1) it is possible to get a high number of cells for transplantation; (2) the body’s signals for promoting proliferation and differentiation are generally intact in the adult organism; (3) a stem cell niche clearly exists which allows for exogenous cells to become integrated into the adult hematopoietic system; and (4) delivery of exogenous cells is relatively easy. Stem cell therapy in the CNS is more complicated than in the hematopoietic system for many reasons. The first is the difficulty in obtaining sufficient numbers of stem cells for transplantation. As mentioned above, stem cells exist in low numbers and in discrete locations in the CNS. Because neurogenesis is limited in the adult CNS, the signals driving NSC proliferation and differentiation are generally absent in the adult; thus the CNS lacks the stem cell “niches” which underlie the success of HSC therapy. Delivery of cells is also more complicated than in the hematopoietic system because cells must be transplanted directly into the site of cell replacement, as they cannot cross the blood– brain bar-

Transgenic animals Dye, DAPI, BrdU, etc. Retroviral, adenoviral Allelic differences Species-specific antibodies X-Y chromosome Magnetic dendrimers NaI symporter Expression of DAT transporter

rier or reliably migrate to the proper site of integration in sufficient numbers. Although cell migration to a site of injury has been noted in several studies, there has been no evidence that cells will migrate to a stem cell niche that might promote survival and proliferation. Another difficulty is the large number of neuronal phenotypes present in the adult brain, making replacement of a specific cell phenotype difficult to engineer. Unlike HSCs, in which the most primitive population is the best source of new cells, NSCs appear to survive and integrate better when a more specific approach is used (Fig. 4). Specificity is achieved through (1) isolation of cells from an appropriate brain region and (2) selection of a defined subpopulation of cells (using the techniques described above). The importance of brain region of origin is demonstrated by transplantation studies in animal models of Parkinson’s disease, in which cells derived from fetal midbrain precursor cells are more effective at integrating into host tissue than ES cells [39]. In either case, source cells (either from the CNS or from differentiated ES cells) are removed from the donor and expanded in vitro using growth factors

Fig. 5. Species-specific primers can distinguish between mouse and human cells, as demonstrated in this RT-PCR of RNA isolated from human ES cells (left) and mouse primary fibroblasts (right) using primers for GATA-2 and GATA-4.

26

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27

Table 6 Antibodies specific to human cells Marker Human specific AC133/2 Human specific Human specific Human specific Human specific NUMA Human specific Human specific Human specific

nestin GFAP CD44 NCAM mitochondria nuclear antigen HLA markers p75

Cell recognized

Source

Ab type

Dividing cells Subset of stem cells Astrocyte precursors Astrocyte precursors Neuronal precursors All cells All cells All cells Predominantly glia Schwann cells

Dr. Messam MACS Sternberger Accurate Chemicon Chemicon Chemicon Chemicon Becton–Dickinson Becton–Dickinson

Rb and monoclonal Labeled Monoclonal Monoclonal IgG Monoclonal Monoclonal Monoclonal Various Monoclonal

to increase the number of cells available for transplantation. Following expansion, it is generally a good idea to re-test the composition of the culture and, if necessary, re-sort for the desired cell population, as even cells grown as neurospheres will often contain both stem cells and more differentiated cells, such as lineage-restricted progenitors. Following selection of the final population, cells can be transplanted into the host. Following transplantation, there is the issue of tracking the cells to determine whether the cells have (1) reached an appropriate destination and (2) differentiated into a functional cell of the appropriate phenotype. Transplanted cells can be tracked in vivo using a number of methods (Table 5). Donor cells can be marked with a dye (such as DAPI) or a retrovirus (or adenovirus) prior to transplantation, allowing the cells to be identified post-transplantation. Transgenic animals expressing a reporter protein (such as ␤-galactosidase or GFP) are good donors because their cells are readily identified in host tissue. Another approach is to use the intrinsic differences between species to identify donor cells. For example, species-specific antibodies and PCR primers can distinguish between different species (Fig. 5). There are a number of human-specific antibodies which are useful for these types of studies (Table 6). Cells can also be identified when transplanted into an animal of the same species but opposite sex on the basis of X-Y chromosomes. The drawback to all of these approaches is the need to sacrifice the animal in order to remove the tissue of interest for analysis; obviously, this approach cannot be used for studying the efficacy of neural transplantation in humans. Less invasive methods for tracking stem cells in vivo include the use of magnetic dendrimers [37]; monitoring radioactive iodide uptake by the sodium iodide symporter using PET [38]; and following expression of the DAT transporter in transplanted cells using radioactively labeled dopamine.

cells because of the variable sources of cells, the variety of stem cells described, and our ability to maintain neural stem cells in culture for prolonged time periods. Fundamental differences in the degree of ongoing cell replacement, the presence or lack of positional cues, and the anatomical differences between marrow and the brain make translating strategies that have worked well for HSC therapy difficult to apply to neural disorders. Nevertheless, some basic similarities bode well for eventual success. FACS sorting is likely to work to select neural cell populations, analogous markers have been identified, and similar positive, negative, and universal stem cell marker strategies appear to work (with important differences). To ensure success, however, one will have to tailor the selection strategy to the source of stem cells and the type of stem cell one wishes to isolate. A rigorous evaluation of all selection strategies in independent laboratories will permit the development of a series of selection protocols that will allow prospective identification of stem cells from any source.

Conclusion

References

Selecting cells for neural cell replacement strategies is intrinsically different from selecting hematopoietic stem

Acknowledgments This work was supported by the NIA, the ALS Center, and the CNS Foundation. The authors thank the members of our laboratory for stimulating discussions and constant support. We also thank Lance A. Edwards for the artistic contribution of Fig. 1. MSR acknowledges the contributions of Dr. S. Rao that made possible the projects underlying the presented work. This paper is based on a presentation at a Focused Workshop on “Stem Cell Plasticity” held in Providence, Rhode Island, April 8 –11, 2003, sponsored by The Leukemia & Lymphoma Society, Roger Williams Medical Center, and the University of Nevada, Reno.

[1] L.O. Jacobson, E.K. Marks, E.O. Gaston, R.E. Zirkle, Effect of spleen protection on mortality following x-irradiation, J. Lab. Clin. Med. 34 (1949) 1538 –1543.

J. Cai et al. / Blood Cells, Molecules, and Diseases 31 (2003) 18 –27 [2] L.O. Jacobson, E.L. Simmons, E.K. Marks, J.H. Eldredge, Recovery from radiation injury, Science 113 (1951) 510 –511. [3] E. Lorenz, D.E. Uphoff, T.R. Reid, E. Shelton, Modification of acute radiation injury in mice and guinea pigs by bone marrow injection, Radiology 58 (1951) 863– 877. [4] C.V. Borlongan, P.R. Sanberg, Neural transplantation for treatment of Parkinson’s disease, Drug Discov. Today 7 (2002) 674 – 682. [5] V.A. McLin, A.M. Zorn, Organogenesis: making pancreas from liver, Curr. Biol. 13 (2003) R96 –R98. [6] A.J. Wagers, J.L. Christensen, I.L. Weissman, Cell fate determination from stem cells, Gene Ther. 9 (2002) 606 – 612. [7] M. Kondo, A.J. Wagers, M.G. Manz, S.S. Prohaska, D.C. Scherer, G.F. Beilhack, J.A. Shizuru, I.L. Weissman, Biology of hematopoietic stem cells and progenitors: implications for clinical application, Annu. Rev. Immunol. 21 (2003) 759 – 806. [8] P.J. Quesenberry, G.A. Colvin, J.F. Lambert, The chiaroscuro stem cell: a unified stem cell theory, Blood 100 (2002) 4266 – 4271. [9] A.J. Wagers, R.I. Sherwood, J.L. Christensen, I.L. Weissman, Little evidence for developmental plasticity of adult hematopoietic stem cells, Science 297 (2002) 2256 –2259. [10] J.L. Christensen, I.L. Weissman, Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells, Proc. Natl. Acad. Sci. USA 98 (2001) 14541–14546. [11] M. Osawa, K. Hanada, H. Hamada, H. Nakauchi, Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell, Science 273 (1996) 242–245. [12] R.J. Jones, J.E. Wagner, P. Celano, M.S. Zicha, S.J. Sharkis, Separation of pluripotent haematopoietic stem cells from spleen colonyforming cells, Nature 347 (1990) 188 –189. [13] W.H. Fleming, E.J. Alpern, N. Uchida, K. Ikuta, G.J. Spangrude, I.L. Weissman, Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells, J. Cell Biol. 122 (1993) 897–902. [14] A.H. Mulder, J.W. Visser, Separation and functional analysis of bone marrow cells separated by rhodamine-123 fluorescence, Exp. Hematol. 15 (1987) 99 –104. [15] G.J. Spangrude, G.R. Johnson, Resting and activated subsets of mouse multipotent hematopoietic stem cells, Proc. Natl. Acad. Sci. USA 87 (1990) 7433–7437. [16] M.A. Goodell, K. Brose, G. Paradis, A.S. Conner, R.C. Mulligan, Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo, J. Exp. Med. 183 (1996) 1797–1806. [17] Y. Liu, Y. Wu, J.C. Lee, H. Xuc, L.H. Pevny, Z. Kaprielian, M.S. Rao, Oligodendrocyte and astrocyte development in rodents: an in situ and immunohistological analysis during embryonic development, Glia 40 (2002) 25– 43. [18] Pevny, L.H., Rao, M.S. Taming the stem cell menagerie, Trends Neurosci. in press (2003). [19] R.M. Seaberg, D. van der Kooy, Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors, J. Neurosci. 22 (2002) 1784 – 1793. [20] S.S. Han, D.Y. Kang, T. Mujtaba, M.S. Rao, I. Fischer, Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord, Exp. Neurol. 177 (2002) 360 –375. [21] L.H. Pevny, S. Sockanathan, M. Placzek, R. Lovell-Badge, A role for SOX1 in neural determination, Development 125 (1998) 1967–1978. [22] J. Cai, Y. Wu, T. Mirua, J.L. Pierce, M.T. Lucero, K.H. Albertine, G.J. Spangrude, M.S. Rao, Properties of a fetal multipotent neural stem cell (NEP cell), Dev. Biol. 251 (2002) 221–240. [23] A. Capela, S. Temple, LeX/ssea-1 is expressed by adult mouse CNS stem cells, identifying them as nonependymal, Neuron 35 (2002) 865– 875.

27

[24] D. Maric, I. Maric, Y.H. Chang, J.L. Barker, Prospective cell sorting of embryonic rat neural stem cells and neuronal and glial progenitors reveals selective effects of basic fibroblast growth factor and epidermal growth factor on self-renewal and differentiation, J. Neurosci. 23 (2003) 240 –251. [25] R.L. Rietze, H. Valcanis, G.F. Brooker, T. Thomas, A.K. Voss, P.F. Bartlett, Purification of a pluripotent neural stem cell from the adult mouse brain, Nature 412 (2001) 736 –739. [26] T. Leemhuis, M.C. Yoder, S. Grigsby, B. Aguero, P. Eder, E.F. Srour, Isolation of primitive human bone marrow hematopoietic progenitor cells using Hoechst 33342 and rhodamine 123, Exp. Hematol. 24 (1996) 1215–1224. [27] S.J. Morrison, K.R. Prowse, P. Ho, I.L. Weissman, Telomerase activity in hematopoietic cells is associated with self-renewal potential, Immunity 5 (1996) 207–216. [28] W. Klapper, T. Shin, M.P. Mattson, Differential regulation of telomerase activity and TERT expression during brain development in mice, J. Neurosci. Res. 64 (2001) 252–260. [29] J. Moreb, J.R. Zucali, Y. Zhang, M.O. Colvin, M.A. Gross, Role of aldehyde dehydrogenase in the protection of hematopoietic progenitor cells from 4-hydroperoxycyclophosphamide by interleukin 1 beta and tumor necrosis factor, Cancer Res. 52 (1992) 1770 –1774. [30] J. Wang, Z. Chen, X. Xia, D. Lu, J. Xue, C. Ruan, A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34⫹ cells to improve combination chemotherapy tolerance, Chin. Med. J. (Engl.) 114 (2001) 25–29. [31] R.W. Storms, A.P. Trujillo, J.B. Springer, L. Shah, O.M. Colvin, S.M. Ludeman, C. Smith, Isolation of primitive human hematopoietic progenitors on the basis of aldehyde dehydrogenase activity, Proc. Natl. Acad. Sci. USA 96 (1999) 9118 –9123. [32] M.B. Kastan, E. Schlaffer, J.E. Russo, O.M. Colvin, C.I. Civin, J. Hilton, Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells, Blood 75 (1990) 1947– 1950. [33] R.J. Jones, J.P. Barber, M.S. Vala, M.I. Collector, S.H. Kaufmann, S.M. Ludeman, O.M. Colvin, J. Hilton, Assessment of aldehyde dehydrogenase in viable cells, Blood 85 (1995) 2742–2746. [34] H. Link, L. Arseniev, O. Bahre, J.G. Kadar, H. Diedrich, H. Poliwoda, Transplantation of allogeneic CD34⫹ blood cells, Blood 87 (1996) 4903– 4909. [35] C.I. Civin, T. Trischmann, N.S. Kadan, J. Davis, S. Noga, K. Cohen, B. Duffy, I. Groenewegen, J. Wiley, P. Law, A. Hardwick, F. Oldham, A. Gee, Highly purified CD34-positive cells reconstitute hematopoiesis, J. Clin. Oncol. 14 (1996) 2224 –2233. [36] A. Urbano-Ispizua, C. Rozman, C. Martinez, P. Marin, J. Briones, M. Rovira, P. Feliz, M.C. Viguria, A. Merino, J. Sierra, R. Mazzara, E. Carreras, E. Montserrat, Rapid engraftment without significant graftversus-host disease after allogeneic transplantation of CD34⫹ selected cells from peripheral blood, Blood 89 (1997) 3967–3973. [37] E.C. Wiener, M.W. Brechbiel, H. Brothers, R.L. Magin, O.A. Gansow, D.A. Tomalia, P.C. Lauterbur, Dendrimer-based metal chelates: a new class of magnetic resonance imaging contrast agents, Magn. Reson. 31 (1994) 1– 8. [38] V. Sharma, G.D. Luker, D. Piwnica-Worms, Molecular imaging of gene expression and protein function in vivo with PET and SPECT, J. Magn. Reson. Imaging 16 (2002) 336 –351. [39] J.H. Kim, J.M. Auerbach, J.A. Rodriquez-Gomez, I. Velasco, D. Gavin, N. Lumelsky, S.H. Lee, J. Nguyen, R. Sanchez-Pernuate, K. Bankiewicz, R. McKay, Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson’s disease, Nature 418 (2002) 50 –56.