C O R R E S P O N D E N C E

medicine approach is the ability toepigenetically drive stem celldifferentiation by ex vivo regulation of thelocal cellular environment rather than bygenetic alteration. Modulation of themicroenvironment would help provide theframework for multipotent cells torecapitulate tissue growth andorganogenesis in a postnatal setting,circumvent the risks of exogenous genetransfer, and may lead to a multi-facetedand cost-effective approach with enormoustranslational implications.

The notion of microenvironmentsaffecting stem cell division and function isnot new. Schofield dubbed these ‘niches’with respect to hematopoietic stem cells4,and subsequent reports have describedtheir presence in numerous tissuesincluding neural, germline, skin, intestinaland others5. The forces driving stem celldifferentiation, or maintaining them in astate of suspended undifferentiation,include secreted and bound messengers orhoming signals. Just as important are thecellular environmental sensors sensitive tooxygen, temperature, chemical gradients,mechanical forces and others cues in themicroenvironment.

In vivo fine tuning of themicroenvironment is typically not anoption; however, the technology torecapitulate many of the key micro-environmental components in vitro isreadily available. Doing so may obviate the

804 VOLUME 22 NUMBER 7 JULY 2004 NATURE BIOTECHNOLOGY

To the editor:We read with interest Roy and colleagues’report1 regarding fetal neural progenitorimmortalization by retroviraloverexpression of telomerase reversetranscriptase. By providing evidence of theefficacy (and, perhaps, relative safety) ofoverexpressing genes that are ‘not foreign’to target cells, their findings are certainly astep forward in the quest to realize thetherapeutic benefit of stem cells. We wereconcerned, however, by Rothstein andSnyder’s2 suggestion in the accompanyingNews & Views Feature that the risks ofgene-based methodology are a ‘myth.’Furthermore, although they ask theimportant question of “what is the mostefficacious yet safest method forexpanding” stem cells ex vivo to overcomethe tremendous biomedical burden of“isolation, expansion…and directeddifferentiation,” we believe this issuedeserves further examination.

The risks of virus-associated genetransfer, although low, are not imaginary(given the evidence of secondarymalignancy in children after stem celltransplantation)3. It is also clear that thelimited availability of embryonic stem (ES)cell sources and the uncertainties regardingthe safety of therapeutic viral-based genetransfer have generated interest inalternative approaches, including the use ofsomatic stem cells. We believe that a keyingredient for implementing a regenerative

Stem cell differentiation

by the Canadian Parliament. Harvard placedCanada in a unique position compared withother developed countries, including theUnited States, Europe, Australia, Japan andKorea, where higher life forms are consideredpatentable.

The decision in Schmeiser is of interestbecause higher life forms are not patentablein Canada, nor are there claims to a plant inthe Monsanto patent. However, Schmeiserwas found to infringe Monsanto’s patent bygrowing glyphosate-resistant canola. TheSupreme Court in Schmeiser confirmed thatgrowing a transgenic plant expressing anucleotide sequence encoding 5-enol-pyruvylshikimate-3-phosphate (ESPS)infringes claims directed to a plant cellcomprising the nucleotide sequence, or thenucleotide sequence itself. The trial judge,had previously concluded “that by growingseed known to be Roundup-tolerant andselling the harvested seed, the defendantsmade use of the invention withoutpermission of the plaintiffs and infringedclaims 1, 2, 5 and 6 of the patent, respectingthe plant gene and claims 22, 23, 27, 28 and45 respecting the plant cells claimed underthe patent”4. The Court of Appeal agreed,stating,“the essence of each claim was thepresence of the Monsanto gene”5. Thisposition has now been approved by theSupreme Court: “…the patented genes andcells are not merely a ‘part’ of the plant;rather, the patented genes are presentthroughout the genetically modified plantand the patented cells compose its entirephysical structure”6.

The finding reached in Schmeiser thatclaims to a cell or gene may confer protectionto a plant is not without precedent as thisprinciple is echoed in Articles 8 and 9 of theEuropean Biotech Directive7. What is unique,however, is the result achieved by combiningHarvard and Schmeiser, that even thoughhigher life forms, such as plants or animalsare not patentable in Canada, a transgenichigher life form may be protectable.

It is likely that a plant or animal producedby breeding alone could not be protected bypatent, as no claims to a cell or gene could beobtained. A transgenic cell, or a cellproduced, for example, through protoplastfusion and comprising a novel property maybe patentable, and protection for the higherlife form may be obtained as a result of thehigher life form comprising the novel cell.

In a recent survey of the Canadian biotechindustry, Ernst & Young8 reported that of the470 biotech companies in Canada,approximately two-thirds have fewer than 20employees, and one-third have fewer than 5

employees. The majority of Canadian biotechis made up of small companies, and a keyrequirement for this emerging sector isstrong protection of innovation. The finaldecision brought down by the SupremeCourt, although important for a giant—Monsanto—has its most importantimplications for small Canadian biotechs.

Without a positive position on the patentprotection of biotech subject matter inCanada, small biotech would suffer, andpossibly even leave. Thus, although it mayappear on the surface as a triumph for bigbusiness, the Schmeiser decision is muchmore. It provides support to Canada’semerging biotech sector. Having someprotection available in Canada for higher lifeforms is a step in the right direction, butfurther developments by the Canadianparliament are still required.

1. Monsanto Canada Inc. v. Schmeiser, 2004 SCC 34;File No. 29437; May 21, (2004).

2. Harvard College v. Canada (Commissioner of Patents),2002 SCC 76; File No. 28155; December 5, (2002).

3. Harvard College v. Canada (Commissioner of Patents),2002 SCC 76; File No. 28155; December 5, (2002),para. [155].

4. 12 C.P.R (4th) 204 (Monsanto Canada Inc. v.Schmeiser; FCTD).

5. Schmeiser v. Monsanto Canada Inc. 2002 FCA 309;File No. A-367-01; September 4, (2002).

6. Monsanto Canada Inc. v. Schmeiser, 2004 SCC 34;File No. 29437; May 21, (2004); para. 42.

7. The European Parliament and the Council of theEuropean Union. Off. J. Eur. Union. L171, 114(1999).

8. Ernst & Young. Resurgence: The AmericasPerspective. Global Biotechnology Report 2004 (May12, 2004).

Konrad Sechley

Gowling Lafleur Henderson LLP, 160 ElginStreet, Suite 2600, Ottawa, Ontario, CanadaK1P 1C3.e-mail: konrad.sechley@gowlings.com

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy

C O R R E S P O N D E N C E

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 7 JULY 2004 805

need to artificially introduce geneticchanges in target cells through genetherapy and circumvent the need to usevirus-mediated gene transfer and itsassociated risks. By avoiding gene therapy,we avoid this risk, perhaps makingapproval of an epigenetic therapeuticapproach by the Food and DrugAdministration potentiallyeasier. Furthermore, atransient change in themicroenvironment canpotentially effect a stable orpermanent change in thedifferentiation process. Theprofound effects of maternaldiet on fetal developmentprovide a glimpse of howimportant changes in themicroenvironment can beon the programming ofdifferentiating cells.

Perhaps the most robustexample of a microenvironmentalregulator of cell fate is oxygen. Althoughthe significance of breathing ambient air(containing ∼ 21% oxygen) to our survivalis obvious, it has been established thatphysiologic ‘normoxia’ is much lower6.Therefore, the traditional paradigm ofculturing cells in humidified ambient air(21% oxygen) may not be optimal formaintaining certain cell types, includingstem cells. Oxygen reduction to 3%–6%promotes the survival of both peripheraland central nervous system stem cells, andcan influence their fate by enhancingcatecholaminergic differentiation7,8.Similarly, human hematopoietic stem cellsdemonstrate increased self-renewal andbone marrow repopulating capability afterhypoxic treatment9. Furthermore, becausecartilage is a relatively avascular tissue,chondrocytes are bathed in a naturallyhypoxic milieu and rely on hypoxia-induced signaling for survival10. Murinemarrow-derived mesenchymal stem cellsundergo enhanced osteochondrogenicdifferentiation in the setting of chronichypoxia, perhaps by returning them to amore ‘natural’ oxygen environment11. Incontrast, preadipocytes do not thrive underhypoxia, and the absence of oxygen triggersa hypoxia inducible factor (HIF)-1αresponse that represses genes essential toadipogenic differentiation12.

Thus, although reduced-oxygenincubation may promote the in vitroexpansion and/or differentiation of manycell types by mimicking in vivo oxygenlevels, it is possible to inhibit the

mesenchymal progenitor cells fromnonconventional means such as suction-assisted lipectomy, and as human trials inwhich reengineered mesenchymal tissuesare introduced, it will become increasinglyimportant to manipulate the growth anddifferentiation of these cells ex vivo. Thus,in addition to the significant role ofexogenous gene transfer (e.g., toimmortalize progenitor cells or rescuespecific genetic defects), we believe that abetter understanding ofmicroenvironmental cues in regulating cellfate also has a ‘niche’ in realizing thetherapeutic potential of stem cells.

1. Roy, N.S. et al. Nat. Biotechnol. 22, 297–305(2004).

2. Rothstein, J.D. & Snyder, E.Y. Nat. Biotechnol 22,283–285 (2004).

3. Hacein-Bey-Abina, S. et al. Science 302, 415–419(2003).

4. Schofield, R. Blood Cells 4, 7–25 (1978).5. Spradling, A., Drummond-Barbosa, D. & Kai, T.

Nature 414, 98–104 (2001).6. Cooper, P.D., Burt, A.M. & Wilson, J.N. Nature

182, 1508–1509 (1958).7. Studer, L. et al. J. Neurosci. 20, 7377–7383

(2000).8. Morrison, S.J. et al. J. Neurosci. 20, 7370–7376

(2000).9. Danet, G.H., Pan, Y., Luongo, J.L., Bonnet, D.A. &

Simon, M.C. J. Clin. Invest. 112, 126–135(2003).

10. Schipani, E. et al. Genes Dev. 15, 2865–2876(2001).

11. Lennon, D.P., Edmison, J.M. & Caplan, A.I. J. CellPhysiol. 187, 345–355 (2001).

12. Yun, Z., Maecker, H.L., Johnson, R.S. & Giaccia,A.J. Dev. Cell 2, 331–341 (2002).

13. Rosenthal, N. N. Engl. J. Med. 349, 267–274(2003).

14. Zuk, P.A. et al. Mol. Biol. Cell 13, 4279–4295(2002).

Ali Salim1,2, Amato J. Giaccia2, and MichaelT. Longaker1

The Departments of Surgery1 and RadiationOncology2, Stanford University School ofMedicine, 257 Campus Drive, Stanford, CA94305, USA.e-mail: Longaker@stanford.edu

Jeffrey Rothstein and Evan Snyderrespond:We are in agreement with Salim and col-leagues that, before contemplating clinicaltranslation of cellular therapies, we mustbetter understand the microenvironment(the ‘niche’) within which these exogenouscells will reside and the dynamic cross-talkthat inevitably continues long after theirimplantation. This, in fact, is one of themessages of our News and Views Feature1

(as well as a theme of our research2–5). Stemcell researchers ignore this cross-talk attheir peril; embracing it and harnessing itwill likely be key to functional reconstitu-

differentiation of others by exposing themto reduced oxygen. But what cells willprovide the ‘raw materials’ for thisapproach? Although the potential of EScells is enormous, ethical and politicalissues currently impede their use13. Issuessurrounding the rights of the unborn fetus,and subsequent government regulation and

limitations on availabilityand applicability ofembryonic tissue, have putthe brakes on whatappeared to be a rapidlyapproaching clinical reality.In contrast, adult-derivedprecursors potentiallyprovide ample quantities ofan autologous source ofregenerative tissue withoutthese ethical and politicalissues13.

Althoughdemonstrations of bone

marrow–derived mesenchymal stem cellplasticity have been reported (anddebated), widespread use of adult-derivedtissue will likely require a relativelypainless, convenient and safe procurementmethod. Some have suggested that the skinfulfill this role. Other reports have begun toemerge suggesting that adipose tissue—which is electively aspirated in largequantities throughout the country eachyear—provides a readily availableautologous source14. Like bone marrow,adipose is supported by a stroma whoseisolation yields a significant amount ofcells capable of osteogenic, adipogenic,neurogenic, myogenic and chondrogenicdifferentiation14. Given that adipose-derived mesenchymal precursors can beharvested in abundant quantities underlocal anesthesia with little patientdiscomfort, we believe they may emerge asan important source for cell-based therapy.One can envision a scenario in which amesenchymal cell fraction is purified froma patient’s bone marrow or liposuctionaspirate, exposed to oxygen and otherenvironmental conditions optimized fordifferentiation along a certain lineage, andultimately returned to the same patient tofill a tissue defect.

In summary, we submit thatunderstanding interactions betweenmicroenvironmental elements andintracellular signaling mechanisms may becrucial to harnessing the plasticity ofmultipotent cells and realizing their utilityin tissue engineering. As it becomestechnically more feasible to acquire

©20

04 N

atur

e P

ublis

hing

Gro

up

http

://w

ww

.nat

ure.

com

/nat

ureb

iote

chno

logy