The document summarizes several studies on stem cell niches. It finds that bone marrow niches for hematopoietic stem cells are regulated by osteoblasts lining bone surfaces. Deleting a BMP receptor in osteoblasts leads to more osteoblasts and more stem cells. Treating with parathyroid hormone also expands the osteoblast population and stem cell numbers by increasing Notch signaling from osteoblasts to stem cells. The microenvironment provided by osteoblasts, including cell-cell signaling via Notch ligands, plays a key role in regulating the stem cell niche in bone marrow.

1. Stem cells find their niche ALLAN SPRADLING, DANIELA DRUMMOND-BARBOSA & TOSHIE KAI

2. Concept of Niches Stable custom environment for stem cells Niches are subsets of tissues and extracellular subsets that can indefinitely house one or more stem cells and control their self-renewal and progeny production in vivo Many niches have one or more specialized cell groups like the skin, GI tract Extracellular matrix and adhesion molecules (basement membrane) may help secure the niches spacially and may modulate the concentration of adhesive and other signalling molecules

3. Figure 1 Niche structure. Niche cells (green) underlying a basement membrane signal to stem cells (red) to block differentiation and regulate division. When a lineage mechanism prevails (lower mitotic cell), the stem cell divides such that one daughter retains its connections to the niche, while the other (yellow) becomes untethered and begins to differentiate. When a population mechanism prevails (upper mitotic cell), stem cell division may be either symmetric (shown) or asymmetric (not shown), as determined by local factors. ECM, extracellular matrix.

4. Figure 2 Manipulating niches. a , Replacement assay to identify niches. Labelled stem cells (blue) are introduced into a tissue. If one or more of the introduced cells becomes associated stably with a particular tissue region and subsequently functions as a stem cell, then the existence and location of a stem cell niche (green) is inferred. b , Cell marking experiments. Marking a single stem cell in vivo (blue), and following the number and location of marked progeny cells reveals its regulatory behaviour. When the stem cell follows a lineage mechanism, the number of labelled stem cells remains relatively constant over many stem cell cycles. When the initially labelled stem cell follows a population mechanism (bottom), initially mosaic niches will sort out stochastically into niches that have all labelled or all unlabelled cells.

5. Figure 3 Male germ cell niches. a , A cross-section of part of a seminiferous tubule of a mouse testis is shown to illustrate the approximate location and properties of the mammalian germline stem cell niche. The stem cells also known as A single (A s ) cells (red) constitute a minority of the basal germ cells in contact with the basement membrane (green). Most other basal cells (yellow) are part of growing germ cell cysts, whose members are interconnected by ring canals. As development proceeds, the cysts leave the basement membrane and move in a highly ordered manner towards the lumen of the tubule (top). All developing germ cells are surrounded by Sertoli cells (green). It is not known whether the region of the Sertoli cell or basement membrane near the stem cells are specialized (dark green). b , A sagittal section of the distal tip of the Drosophila testis is drawn schematically and leaves out most of the cells for clarity. The hub cells (green) are attached to 5–9 groups comprising one germline stem cell and two cyst progenitor stem cells (red). The basement membrane (green) may be thinner and specialized (dark green) near the hub. Germline stem cell daughters called gonialblasts are encased by two cyst cells, begin to differentiate, and move posteriorly away from the hub. After four more divisions, the cyst cells (yellow) cease division and synchronously enter meiosis.

6. Figure 5 Epithelial stem cell niches. a , Mammalian epidermal stem cells. A hair follicle and a segment of adjacent skin is illustrated. The dermal papilla (DP) signals to matrix stem cells (red) located across a basement membrane (green). Matrix cell daughters (yellow) differentiate into a variety of cell types, including the medulla, cortex and cuticle of the hair shaft (brown), the inner root sheath (IRS) and the outer root sheath (ORS). About two-thirds of the way up an anagen follicle lies the bulge — an expanded region that contains long-term stem cells (red). These cells periodically replenish (arrows) the matrix cells, and also help maintain the sebaceous gland (SG) and the epidermal stem cells (red, top layer) that lie against the basement membrane (not shown) overlying the basal layer in interfollicular regions. b , A mammalian gut crypt is a tube of cells arrayed on a basement membrane (green). Stem cells (red) are located in the basal region along with Paneth cells, but their exact location is variable and both types account for only a fraction of the cells present in the regions shown. A portion of the basement membrane in the stem cell region may be specialized (dark green). Stem cell progeny (yellow) known as transit amplifying cells (TA) move upwards and differentiate. Underlying mesenchymal cells (green) send signals that help regulate stem cell activity. Dermal papilla produce FGF-7, BMP-4, KGF Inner root sheath Outer root sheath transit amplifying cells

7. hsc msc osetoid osteoblasts Blood cells Role of osteoblasts and hematopoiesis Osteobalst output 0.01% of blood cells (350 bill/day) Stromal cell cultures express OB markers OB express hematopoietic cytokines

9. Bmpr1a/b signaling: negative on osteoblasts,MSC proliferation Bmpr1a -/- embryonic lethal with major defects in blood formation Authors use conditional KO of Bmpr1a: Mating Bmpr1a fx/fx with Mx1-Cre (poly IC induction) Also used triple genotype mice bearing Mx1-Cre, Bmpr1a fx/fx, and Z/EG alleles

11. ST LT Due to decreased BM cavity Transplanted (2 x 103) with WT (ly 5.1) into control vs Brmp1a mutants Transplanted (2 x 103) with Brmp1a (ly 5.2) into control vs WT recipients (ly5.1) It is the microenvironment In competitive repop studies using whole BM, Bmpr1a wins out 2:1 (data not shown)

12. TBLA

13. Poly IC induction

15. Triple geneotype mice (correct targeting to Bmpr1a region)

17. # N-Cadherin+ Osteoblasts (per section) 78% 33%

18. SNO: spindle shpaed osteoblast N-cathedrin+ cells

19. Possible mechanisms Intrinsic changes in stem cells that either promote self renewal or decreased apoptosis Internal defect in differentation External influence of microenvironment**** Wild type to Bmpr1a transplantation (3 mo 1.6-2.0 x WT donor) Ectopic Trabecular bone-like area Increased # of HSCs in TBLA No differnce in MSC per femur (data not shown) 10X increased bone volume 3 x # osteoblasts Same # osteoclasts

20. Osteoblastic cells regulate the haematopoietic stem cell niche L. M. CALVI 1,* , G. B. ADAMS 3,* , K. W. WEIBRECHT 3 , J. M. WEBER 1 , D. P. OLSON 3 , M. C. KNIGHT 4 , R. P. MARTIN 3 , E. SCHIPANI 4 , P. DIVIETI 4 , F. R. BRINGHURST 4 , L. A. MILNER 2 , H. M. KRONENBERG 4 & D. T. SCADDEN 3 Nature 425:841

21. Figure 1 Nature 425:841 BMT-male PTH+inreased a Ability to reconstitute female WT By real time DNA PCR for Y

22. Figure 2 Nature 425:841 Stromal cells from from tg (n=6) And wt (n=6) improves support Of LTC-IC..no CFU data Increased Jag-1 in tg Increased jag/osteopontin Increased NICD In tg lin-Sca+c-Kit+ HSC expansion mediated By stromal cells Gamma-secretase of notch

23. Figure 3 Nature 425:841 When WT stroma is grown in the presence of PTH, the Effect same as tg stroma No effect of PTH alone Increased # alk phos OB Effect blocked by Gamma-secretase inhibitor

24. Figure 4a Nature 425:841 PTH was administered for one month prior to myeloablative BMT and animals were given limiting numbers of donor stem cells

25. Figure 4b-e Nature 425:841 Histology 4 weeks Post-transplant

26. Nature 425:778 Figure 1 Birth control for stem cells. The niche that regulates the birth and differentiation of blood-forming (haematopoietic) stem cells is formed of osteoblasts (a type of bone-marrow cell) that line the inner surface of bone. Zhang et al. 7 showed that depleting osteoblasts of a receptor for bone morphogenetic protein (BMP) caused a doubling in both the osteoblast population and the stem-cell population. Calvi et al. 8 found a parallel expansion of the stem-cell population when they increased the numbers of osteoblasts by using parathyroid hormone (PTH). Lemischka et al Nature 425: 778, 2003